Antibody having activity of inhibiting hepatitus c virus (hcv) infection and use thereof

ABSTRACT

An object of the present invention is to provide an antibody inhibiting infection with hepatitis C virus (HCV). The present invention provides an anti-hepatitis C virus antibody that recognizes a whole or a part of the conformation of a hepatitis C virus particle as an epitope and binds thereto, so as to be able to inhibit the binding of hepatitis C virus to the surface of a host cell and to inhibit HCV infection, a humanized antibody thereof, and an inhibitory agent for infection with hepatitis C virus.

This application is a Divisional of application Ser. No. 13/504,765,filed Apr. 27, 2012, which is a National Phase of PCT InternationalApplication No. PCT/JP2010/069323 filed on Oct. 29, 2010, and whichclaims priority to Patent Application Nos. 2009-251165, filed in Japanon Oct. 30, 2009 and 2009-251341, filed in Japan on Oct. 30, 2009. Theentire contents of all of the above applications are hereby incorporatedby reference.

TECHNICAL FIELD

The present invention relates to an antibody having activity ofinhibiting hepatitis C virus (HCV) infection and use thereof.

BACKGROUND ART

The hepatitis C virus (which may be abbreviated as “HCV” hereinafter) isan RNA virus that is classified as a member of the genus Hepacivirus ofthe family Flaviviridae. It has been identified as a major causativevirus of non-A and non-B hepatitis (non-patent document 1). The HCVgenome encodes a precursor protein that is converted into 10 types ofvirus protein (i.e., Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, andNS5B) via post-translational cleavage by host-derived signal peptidaseor HCV-derived proteases. Of these virus proteins, Core, E1, E2, and p7proteins are classified as structural proteins, and NS2, NS3, NS4A,NS4B, NS5A, and NS5B proteins are classified as non-structural proteins.

HCV is mainly transmitted via blood transfusion. Highly sensitivemethods for detecting HCV have been established today, and the number ofnew HCV patients because of blood transfusion has dramaticallydecreased. However, at present, the number of HCV carriers includingso-called virus carriers who have not yet developed hepatitis symptomsis deduced to be over 2,000,000 in Japan, and is over 170,000,000 in theworld. This is mainly because the rate of chronicity of hepatitis due toHCV infection is as high as 70% to 80% and there are no effectiveantiviral agents other than interferons at present. Further, chronichepatitis C caused by HCV infection would become worse and lead tocirrhosis during the following some 20 years, finally resulting in livercancer. Further, liver cancer is known to result in relapse for manypatients due to inflammation that continuously occurs at noncancerousparts even if cancer is surgically excised.

Therefore, development of antiviral drugs and vaccines with beneficialeffects has been desired for the purpose of preventing virus carriersfrom developing the disease and eliminating viruses. For this purpose,detailed information about the HCV life cycle should be clarified, suchas regarding the ways in which HCV invades, replicates, grows in hostcells, and HCV affects host cells.

The HCV life cycle involves the series of cycles described below. First,HCV binds to a specific protein (virus receptor) on the cell surface andis incorporated by endocytosis into the host cell. Next, HCV genomic RNAis released into the host cytoplasm from viral particles (uncoating).Subsequently, HCV protein precursors encoded by the released HCV genomicRNA are translated. After each virus protein has been generated byprocessing, the HCV genomic RNA is replicated by RNA polymerase, whichis one of the generated virus proteins. The thus replicated HCV genomicRNA is packaged by the Core protein and envelope proteins (E1 proteinand E2 protein), which are structural proteins, so that new viralparticles are formed. Finally, viral particles break the host cellmembranes and are then released from the cells.

Therefore, it is important to develop a method for inhibiting at leastone of the above steps in the process of HCV infection, in order toprevent HCV carriers from developing the disease and to eliminate thevirus.

HCV envelope proteins are considered to play a key role in the bindingof HCV to cell surfaces. Thus, research has been conducted forpreparation of antibodies against envelope proteins in blood serumsamples of HCV patients. However, the percentage of HCV patientsexhibiting positive reactions with either the C100 antibody (theNS4-NS-5 antibody) or the anti-core antibody, both of them or ananti-envelope protein antibody was found to be approximately 10%. Sinceonly about 10% of HCV patients are naturally cured with a neutralizingantibody (non-patent document 2), it is thought that as few as 1% of allpatients who are thought to be cured by the anti-envelope proteinantibody. This is thought to be due to the presence of a mechanism thatinhibits or suppresses the production of antibodies against HCV envelopeproteins (non-patent document 3)

Meanwhile, non-patent document 4 discloses that when one of the HCVenvelope proteins, the E2 protein, is expressed in a mammal, the E2protein specifically binds to CD81 existing on human cell surfaces.Based on the experimental result, isolation of an antibody that exhibitsNOB (neutralization of binding) activity that inhibits the bindingbetween the E2 protein and CD81 from a hepatitis C patient has beenattempted. For example, through construction of an antibody gene libraryfrom the bone-marrow lymphocytes of a chronic hepatitis C patientaffected by HCV of genotype 1a, followed by employment of a phagedisplay method, the above antibody has been isolated (patent document1). Moreover, an antibody exhibiting NOB activity has also been isolatedby a method for preparing hybridomas from peripheral B cells of ahepatitis C patient affected by HCV of genotype 1b (non-patent document5 and patent document 2). However, with methods for preparing monoclonalantibodies from HCV patients, it is difficult to obtain a variety ofrepertoires of infection-inhibiting antibodies and to find antibodiesuseful as anti-HCV agents, since only the patients having HCVinfection-inhibiting antibodies can be used herein. Also, it has beenreported that an antibody exhibiting NOB activity does not alwaysinhibit infection (non-patent document 8).

Furthermore, a method that involves inducing an antibody viaadministration of a recombinant envelope protein to a mouse (patentdocument 3) and a method that involves fusing lymphocytes to myelomacells to prepare antibody-producing hybridomas (thus preparing anantibody against an envelope protein) have been attempted (patentdocument 4 and non-patent document 6). However, no effective antibodyinhibiting HCV infection has been obtained to date. No antibodyneutralizing HCV infection has been prepared by immunizing an animalwith an envelope protein. One of the suggested reasons for this lack isthat a recombinant envelope protein to be used for immunization has astructure differing from that of the virus's original envelope protein.It has also been reported that recombinant envelope proteins tend toaggregate so that they are unable to maintain their originalconformations (non-patent document 7).

Therefore, in view of treatment and prevention using HCV antibodies,development of antibodies against envelope proteins that are capable ofinhibiting viral infection and a new method for effectively inducingsuch antibodies have been desired.

Starting from the above background, technique for preparing infectiousHCV particles with a cell culture system has been recently established(patent documents 5, 6, and 7). Unlike the above method, which involvescausing the expression of a recombinant envelope protein by generecombination techniques and using the resultant as an antigen, HCVparticles prepared using such a cell culture system are infectious, andthus the conformation of the HCV antigen may be maintained.

The conformation of HCV is composed of an envelope comprising envelopeproteins (E1 protein and E2 protein) and a lipid membrane. These E1 andE2 proteins are thought to bind to each other, forming a complex(non-patent document 7).

On the other hand, envelope proteins of an AIDS virus form a trimer. Ithas been revealed that an antibody recognizing the conformation of thetrimer as an epitope (antigenic determinant) is effective against a widerange of AIDS viruses, compared with conventional anti-AIDS virusantibodies, and has high neutralization activity. This suggests that itis important for an antibody with such neutralization activity to beable to recognize the conformation of a viral antigen as an epitope(non-patent document 9).

PRIOR ART DOCUMENTS Patent Documents

-   Patent document 1 JP Patent Publication (Kohyo) No. 2005-531286 A-   Patent document 2 JP Patent Publication (Kohyo) No. 2006-504645A-   Patent document 3 JP Patent Publication (Kohyo) No. 2004-500366 A-   Patent document 4 JP Patent Publication (Kohyo) No. 6-505389 A    (1994)-   Patent document 5 WO05080575A1-   Patent document 6 WO06022422A1-   Patent document 7 WO06096459A2-   Non-patent document 1 Choo et al., Science, 1989, Vol. 244, p.    359-362-   Non-patent document 2 Matsuura et al., J. Virol., 1992, Vol. 66, p.    1425-1431-   Non-patent document 3 Saito et al., Experimental Medicine    (JIKKEN-IGAKU; Japanese), 1991, Vol. 9, p. 2075-2080-   Non-patent document 4 Pileri et al., Science, 1998, Vol. 282, p.    938-941-   Non-patent document 5 Hadlock et al., J. Virol., 2000, Vol. 74, p.    10407-10416-   Non-patent document 6 Suzuki et al., SAISHIN IGAKU (Japanese), 2003,    Vol. 58, p. 2017-2022-   Non-patent document 70p de Beeck et al., J. Gen. Virol., 2001, Vol.    82, p. 2589-2595-   Non-patent document 8 Burioni et al., J. Virol., 2002, Vol. 76, p.    11775-11779-   Non-patent document 9 Laura M. Walker et al., Science, 2009, Vol.    326, p. 285

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

An object of the present invention is to provide an antibody inhibitingHCV infection.

Means for Solving the Problem

As a result of intensive studies, the present inventors have succeededin obtaining a plurality of monoclonal antibodies having activity ofinhibiting HCV infection from antibody-producing hybridomas preparedfrom mice to which infectious HCV particles have been administered.These monoclonal antibodies are anti-HCV antibodies that recognize theconformation of a complex consisting of the HCV E1 protein and E2protein, as an epitope. Such antibodies capable of recognizing such aconformation as an epitope are predicted to be effective against a widerange of HCV and not to lose the infection-inhibiting capacity due toHCV mutation. The present invention has been completed based on thesefindings, encompassing the following (1) to (24).

(1) An anti-HCV antibody, which recognizes as an epitope theconformation of a complex consisting of an E1 protein and an E2 proteinof HCV particles, and has activity of inhibiting infection with HCV.(2) The anti-HCV antibody according to (1), wherein the amino acidsequences of the E1 protein and the E2 protein contain the amino acidsequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, in thesequence listing.(3) The anti-HCV antibody according to (1) or (2), wherein the HCVparticles are produced from a chimeric HCV genome composed by connectingportions of the genome of the HCV J6CF strain and JFH-1 strain, and thechimeric HCV genome is the following (i) or (ii):(i) a chimeric HCV genome, in which 5′ untranslated region, coreprotein-coding sequence, E1 protein-coding sequence, E2 protein-codingsequence, and p7 protein-coding sequence derived from J6CF strain, NS2protein-coding sequence, NS3 protein-coding sequence, NS4Aprotein-coding sequence, NS4B protein-coding sequence, NS5Aprotein-coding sequence, NS5B protein-coding sequence, and the 3′untranslated region derived from the JFH-1 strain are connected from the5′ side in this order; or(ii) a chimeric HCV genome, in which 5′ untranslated region, coreprotein-coding sequence, E1 protein-coding sequence, E2 protein-codingsequence, p7 protein-coding sequence, and the amino acid sequenceencoding 16^(th) amino acid residues from the N-terminus of an NS2protein-coding region derived from the J6CF strain, amino acid sequencefollowing the 17^(th) amino acid residue from the N-terminus to theC-terminal amino acid residue of the NS2 protein-coding region, NS3protein-coding sequence, NS4A protein-coding sequence, NS4Bprotein-coding sequence, NS5A protein-coding sequence, NS5Bprotein-coding sequence, and the 3′ untranslated region derived from theJFH-1 strain are connected from the 5′ side in this order.(4) The anti-HCV antibody according to (3), wherein the chimeric HCVgenome is a nucleic acid consisting of the nucleotide sequence shown inSEQ ID NO: 2 in the sequence listing (and when the nucleic acid is RNA,thymine (T) in the nucleotide sequence is read as uracil (U)).(5) The anti-HCV antibody according to any one of (1) to (4) above,wherein the activity of inhibiting HCV infection is to inhibit thebinding of an HCV particle to the surface of a host cell.(6) The anti-HCV antibody according to any one of (1) to (5) above,which is produced by a hybridoma cell line deposited under Accession No.FERM BP-11263.(7) The anti-HCV antibody according to any one of (1) to (5) above,which is produced by a hybridoma cell line deposited under Accession No.FERM BP-11264.(8) The anti-HCV antibody according to any one of (1) to (5) above,which is a humanized antibody.(9) A hybridoma cell line, the Accession No. of which is FERM BP-11263.(10) A hybridoma cell line, the Accession No. of which is FERM BP-11264.(11) An inhibitory agent for HCV infection, comprising the anti-HCVantibody according to any one of (1) to (8) above as an activeingredient.(12) The anti-HCV antibody according to any one of (1) to (5) above,comprising a heavy chain variable region that contains a complementaritydetermining region containing the amino acid sequences shown in SEQ IDNOs: 18, 20, and 22 in the sequence listing.(13) The anti-HCV antibody according to (12) above, comprising a heavychain variable region that contains the amino acid sequence shown in SEQID NO: 13 in the sequence listing.(14) The anti-HCV antibody according to (1), (2), (3), (4), (5), (12),or (13) above, comprising a light chain variable region that contains acomplementarity determining region containing the amino acid sequencesshown in SEQ ID NO: 25, 27, and 29 in the sequence listing.(15) The anti-HCV antibody according to (14) above, comprising a lightchain variable region that contains the amino acid sequence shown in SEQID NO: 14 in the sequence listing.(16) The anti-HCV antibody according to any one of (1) to (5) above,comprising a heavy chain variable region that contains a complementaritydetermining region containing the amino acid sequences shown in SEQ IDNOs: 32, 34, and 36 in the sequence listing.(17) The anti-HCV antibody according to (16) above, comprising a heavychain variable region that contains the amino acid sequence shown in SEQID NO: 15 in the sequence listing.(18) The anti-HCV antibody according to (1), (2), (3), (4), (5), (16),or (17) above, having a light chain variable region that contains acomplementarity determining region containing the amino acid sequencesshown in SEQ ID NO: 39, 41, and 43 in the sequence listing.(19) The anti-HCV antibody according to (18) above, having a light chainvariable region that contains the amino acid sequence shown in SEQ IDNO: 16 in the sequence listing.(20) The antibody according to (12) to (19) above, 1 to 5 amino acids ina framework region are deleted, substituted, inserted, or added in theamino acid sequence of the above heavy chain variable region or lightchain variable region.(21) The anti-HCV antibody according to any one of (12) to (20) above,which is a humanized antibody.(22) A fragment of the anti-HCV antibody according to (1), (2), (3),(4), (5), (6), (7), (8), (12), (13), (14), (15), (16), (17), (18), (19),(20), or (21) above, which recognizes as an epitope the conformation ofa complex of the E1 protein and the E2 protein of HCV particles and hasactivity of inhibiting HCV infection.(23) A polynucleotide, encoding the antibody according to (1), (2), (3),(4), (5), (6), (7), (8), (12), (13), (14), (15), (16), (17), (18), (19),(20), or (21) or a fragment of the antibody according to (22) above.(24) A vector containing the polynucleotide according to (23) above.

This description includes the contents of the descriptions and/ordrawings of Japanese Patent Application Nos. 2009-251165 and2009-251341, which are priority documents of the present application.

Effects of the Invention

The antibody having the activity of inhibiting HCV infection of thepresent invention and use thereof can be used for treating or preventinghepatitis C and studies for elucidation of the HCV infection mechanism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the activity of inhibiting HCV infection of IgG fractionsfrom the serum samples of mice to which J6/JFH-1-HCV particles wereadministered.

FIG. 2 shows the activity of inhibiting HCV infection of the P18-9Emonoclonal antibody against J6CF infectious HCV-like particles.

FIG. 3 shows the activity of inhibiting HCV infection of the P19-7Dmonoclonal antibody against J6CF infectious HCV-like particles.

FIG. 4 shows the activity of inhibiting HCV infection of the P18-9Emonoclonal antibody against J6/JFH1 infectious HCV particles.

FIG. 5 shows SEQ ID NOs of the amino acid sequences of complementaritydetermining regions (CDRs) and framework regions (FRs) in the heavychain and light chain variable regions of the P18-9E monoclonal antibodyand the P19-7D monoclonal antibody.

FIG. 6 shows the result of enzyme immunoassay (EIA) for the 8D10-3monoclonal antibody using plates on which the recombinant E1 and E2proteins were immobilized.

FIG. 7 shows the result of EIA for the 8D10-3 monoclonal antibody andthe P18-9E monoclonal antibody using plates on which HCV-like particles(HCV-VLP) were immobilized.

FIG. 8 shows the result of EIA for the 8D10-3 monoclonal antibody andthe P19-7D monoclonal antibody using plates on which HCV-like particles(HCV-VLP) were immobilized.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The embodiments of the present invention are as described in detailbelow. The present invention can be implemented via conventionalmolecular biological and immunological techniques within the technicalscope in the art. Such techniques are thoroughly explained in, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory (Third Edition, 2001) or Ed Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. Anti-HCV Antibody and Fragment Thereof.

An embodiment of the present invention relates to an anti-HCV antibodyor a fragment thereof, which reacts with an HCV particle as an antigenand has activity of inhibiting HCV infection.

1-1. Anti-HCV Antibody and Fragment Thereof.

The term “anti-HCV antibody” in the present invention refers to anantibody that is induced using an HCV particle produced from a chimericHCV genome (described later) as an antigen. Such an anti-HCV antibody isnamely a neutralizing antibody that recognizes and binds to theconformation in HCV particles as an epitope, and has activity ofinhibiting infection of host cells with HCV. Examples of the anti-HCVantibody of the present invention include polyclonal antibodies ormonoclonal antibodies. The anti-HCV antibody is preferably a monoclonalantibody. The term “monoclonal antibody” as used herein refers to apolypeptide that contains a single immunoglobulin, or a framework regionthereof (hereinafter, referred to as “FR”), and a complementaritydetermining region (hereinafter, referred to as “CDR”), and is capableof specifically binding to and recognizing an HCV particle as anantigen. Examples of the known antibody classes of the aboveimmunoglobulin include IgG, IgM, IgA, IgE, and IgD. The antibody of thepresent invention may be of any class. An IgG antibody is preferable.

Specific examples of the anti-HCV antibody of the present inventioninclude antibodies comprising CDRs that contain the amino acid sequencesshown in SEQ ID NOs: 18, 20, and 22, or SEQ ID NOs: 32, 34, and 36 in aheavy chain variable region (H chain V region: hereinafter, referred toas “VH”). An example thereof is an antibody having VH that contains theamino acid sequence shown in SEQ ID NO: 13 or 15.

Also, specific examples of the anti-HCV antibody of the presentinvention include antibodies comprising CDRs that contain the amino acidsequences shown in SEQ ID NOs: 25, 27, and 29, or SEQ ID NO: 39, 41, and43 in a light chain variable region (L chain V region: hereinafter,referred to as “VL”). An example thereof is an antibody having VL thatcontains the amino acid sequence shown in SEQ ID NO: 14 or 16.

The amino acid sequence of the antibody of the present invention, avariable region (hereinafter, referred to as “V region”) of a fragmentthereof (described later), or particularly FR contained in the regionmay contain a mutation as long as it can maintain the activity ofspecifically binding to HCV particles. Specifically, 1 to 5, preferably1 to 4, more preferably 1 to 3, further preferably 1 or 2 amino acids inthe amino acid sequence of FR may be deleted, substituted, inserted, oradded. The reason for this is as follows. Since FR is the regioncomposing the skeleton of a V region and thus is not directly involvedin the antigen-binding specificity of the antibody, the activity ofspecifically binding to HCV particles is highly likely maintained evenwhen the above mutation is introduced into the relevant region. On theother hand, introduction of a mutation into CDR highly likely causes achange in the antigen-specific binding activity, and thus is generallynot preferable. However, there is a known example that introduction of amutation into CDR may significantly enhance the binding activity of anantibody. Therefore, in the present invention, the above mutation may bewithin CDR. In this case, CDR may contain a deletion, a substitution, aninsertion, or an addition of 1 to 3, and preferably 1 or 2 amino acids.For such introduction of a mutation, a phage vector described later canbe used. A phage vector can be conveniently used for screening for anantibody containing a mutation that retains the activity of specificallybinding to HCV particles or enhances such specific activity, since itenables rapid expression of an introduced antibody in a large amount,and is capable of expressing the antibody molecules in sufficientamounts on the host bacterial cell surfaces.

The term “a fragment thereof” in the present invention refers to apartial region of the above anti-HCV antibody that is a polypeptidechain having activity substantially equivalent to the antigen-specificbinding activity of the relevant antibody or a complex thereof. Anexample thereof is an antibody portion containing at least one antigenbinding site, that is, a polypeptide chain having at least one VL and atleast one VH or a complex thereof. Specific examples thereof includemany sufficiently characterized antibody fragments resulting fromcleavage of immunoglobulin with various peptidases. More specificexamples thereof include Fab, F(ab′)_(2′) and Fab′. Fab is a fragmentthat is generated by cleaving an IgG molecule with papain at a positioncloser to the N-terminal side than the position of the disulfide bond inthe hinge region, which is composed of a polypeptide consisting of VHand H chain C region (heavy chain constant region: hereinafter, referredto as “CH”) 1 adjacent to VH among 3 domains (CH1, CH2, and CH3)composing CH and a light chain. F(ab′)₂ is a Fab′ dimer that isgenerated by cleaving an IgG molecule with pepsin at a position closerto the C-terminal side than the position of the disulfide bond in thehinge region. Fab′ has the H chain that is slightly longer than that ofFab since it contains the hinge region, but has a structuresubstantially equivalent to that of Fab (see Fundamental Immunology,Paul ed., 3d ed. 1993). Fab′ can be obtained by reduction of F(ab′)₂under mild conditions to cleave disulfide bond in the hinge region.These antibody fragments contain antigen binding sites, being capable ofspecifically binding to antigens (that is, HCV particles in the presentinvention).

The anti-HCV antibody or a fragment thereof of the present invention canbe modified. Examples of modification mentioned herein include bothfunctional modification required for the antibody or a fragment thereofof the present invention to have activity of specifically binding to HCVparticles (e.g., glycosylation) and labeling required for detection ofthe antibody or a fragment thereof of the present invention. Examples oflabeling of the above antibody include labeling with fluorescent dyes(FITC, rhodamine, Texas Red, Cy3, and Cy5), fluorescent proteins (e.g.,PE, APC, and GFP), enzymes (e.g., horseradish peroxidase, alkalinephosphatase, and glucose oxidase), or biotin or (strepto)avidin. Also,glycosylation of the antibody of the present invention may be modifiedto adjust the affinity of the antibody for a target antigen. Such amodification may be achieved by, for example, changing one or moreglycosylated sites within the sequence of an antibody. This is morespecifically explained as follows. For example, one or more amino acidsubstitutions are introduced into an amino acid sequence composing oneor more glycosylated sites within FR to remove the glycosylated sites,so that the sites can be deglycosylated. Such deglycosylation iseffective for enhancing the affinity of an antibody for an antigen (U.S.Pat. Nos. 5,714,350 and 6,350,861).

The anti-HCV antibody or a fragment thereof of the present inventionpreferably has high affinity, such that the dissociation constantbetween the antibody (or a fragment thereof) and HCV particles is 5.0e⁻⁹ M or less, preferably 1.0 e⁻⁹ M or less, more preferably 5.0 e⁻¹⁰ Mor less, 1.0 e⁻¹⁰ M or less, 5.0 e⁻¹¹M or less, 1.0 e⁻¹¹M or less, 5.0e⁻¹²M or less, or 1.0 e⁻¹²M or less. The dissociation constant can bemeasured using techniques known in the art. For example, dissociationconstant may also be measured using rate assessment kit software of aBIAcore system (GE Healthcare Bioscience). In addition, dissociationconstant is preferably measured in the presence of 0.3M sodium chloridefor precise measurement. Such conditions may be appropriatelydetermined.

The anti-HCV antibody or a fragment thereof of the present invention isan antibody or a fragment thereof derived from an arbitrary organism,and preferably derived from a mammal or a fragment thereof. When theanti-HCV antibody or a fragment thereof of the present invention isadministered to a human for the purpose of inhibiting HCV infection, itis desirably a human antibody, or a recombinant antibody synthesizedchemically or by a recombination DNA method. This is because theconstant region (hereinafter referred to as “C region”) of an anti-HCVantibody derived from a non-human organism has immunogenicity in a humanbody, so that immune reaction is induced against the antibody, and thusthe above purpose cannot be achieved.

The above “recombinant antibody” as used herein refers to a chimericantibody, humanized antibody, or a synthetic antibody, for example.

The term “chimeric antibody” refers to an antibody resulting fromsubstitution of the C region of an antibody with the C region of anotherantibody. An example thereof is an antibody resulting from substitutionof the C region in a mouse monoclonal antibody (P18-9E or P19-7D) havingactivity of inhibiting HCV infection described later with the C regionof a human antibody. This can alleviate immune reaction against theantibody within a human body. A more specific example of a chimericantibody in the present invention is an antibody wherein VL contains theamino acid sequence shown in SEQ ID NO: 14 or 16 derived from ananti-HCV mouse monoclonal antibody and the L chain C region (light chainconstant region: hereinafter, referred to as “CL”) contains an aminoacid sequence in CL of an arbitrary human antibody, and/or VH containsthe amino acid sequence shown in SEQ ID NO: 13 or 15 derived from ananti-HCV mouse monoclonal antibody and CH contains an amino acidsequence in CH of an arbitrary human antibody.

The term “humanized antibody” is also referred to as a reshaped humanantibody, such antibody is a mosaic antibody obtained by grafting CDR ofan antibody of a non-human mammal (e.g., a mouse) into the CDR of ahuman antibody, for example. Mainly CDR groups in the V regions areresponsible for the antigen-binding specificity of antibodies.Therefore, when a recombinant antibody having binding properties similarto those of a specific antibody is prepared, there is no need to obtainthe full-length amino acid sequence of the antibody. Through the use ofan existing recombination DNA technique, a mosaic antibody is preparedby substituting the DNA sequence encoding each CDR region derived fromthe antibody with a DNA sequence encoding a human antibody-derived CDRcorresponding thereto, and then causing the expression of theresultants. Thus, a recombinant antibody with properties of such aspecific antibody can be obtained through simulation thereof. A generalgene recombination technique for preparation of a humanized antibody isalso known (European Patent Application Publication No. EP 125023). Anexample thereof is a method that involves designing a DNA sequence toligate CDR of a mouse antibody against FR of a human antibody and thensynthesizing the DNA sequence by a PCR method using severaloligonucleotides as primers so as to have overlap portions at terminalregions of both CDR and FR. In the present invention, CDRs of anti-HCVmouse monoclonal antibodies (P18-9E and P19-7D) were revealed. Hence, ahumanized antibody can be prepared by the following method, for example.

The term “synthetic antibody” refers to an antibody or an antibodyfragment synthesized chemically or via a recombination DNA method.Examples thereof include a monomeric polypeptide molecule prepared byartificially ligating one or more VLs and one or more VHs of a specificantibody via a linker peptide or the like having an appropriate lengthand sequence and a polymeric polypeptide thereof. Specific examples ofsuch a polypeptide include single-stranded Fv (scFv: single chainFragment of variable region) (see Pierce Catalog and Handbook,1994-1995, Pierce Chemical Co., Rockford, Ill.), a diabody, a triabody,a tetrabody and the like. In an immunoglobulin molecule, VL and VH aregenerally located on different polypeptide chains (light chain and heavychain). Single-stranded Fv is a synthetic antibody fragment having astructure in which these V regions existing on the two polypeptidechains are linked via a flexible linker with a sufficient length, andsaid two V regions are contained in a single polypeptide chain. Both Vregions within the single-stranded Fv can form one functional antigenbinding site through self assembly thereof. Single-stranded Fv can beobtained by incorporating recombinant DNA encoding the Fv into a phagegenome with a known technique, followed by expression thereof. A diabodyis a molecule having a structure based on the dimeric structure ofsingle-stranded Fv (Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A.90: 6444-6448). For example, when the above linker has a length shorterthan about 12 amino acid residues, two variable sites withinsingle-stranded Fv cannot perform self assembly. However, through theformation of a diabody, specifically, through interaction between thetwo single-stranded Fvs, the assembly of the VL of one Fv chain with theVH of the other Fv chain becomes possible, so that two functionalantigen binding sites can be formed (Marvin et al., 2005, ActaPharmacol. Sin. 26:649-658). Moreover, through addition of cysteineresidues to the C-termini of single-stranded Fvs, disulfide bond betweenthe two Fv chains can be formed, so that a stable diabody can also beformed (Olafsen et al., 2004, Prot. Engr. Des. Sel. 17: 21-27). Asdescribed above, a diabody is a divalent antibody fragment. However, theantigen binding sites thereof are not required to bind to the sameepitopes and may have bispecificity so that they recognize differentepitopes for specific binding. For example, one of the antigen bindingsites may be composed of VH that comprises CDRs containing the aminoacid sequences shown in SEQ ID NOs: 18, 20, and 22 (corresponding toCDR1, CDR2, and CDR3, respectively, in VH of P18-9E) and VL thatcomprises CDRs containing the amino acid sequences shown in SEQ ID NOs:25, 27, and 29 (corresponding to CDR1, CDR2, and CDR3, respectively, inVL of P18-9E), and the other antigen binding site may be composed of VHthat comprises CDRs containing the amino acid sequences shown in SEQ IDNOs: 32, 34, and 36 (corresponding to CDR1, CDR2, and CDR3,respectively, in VH of P19-7D) and VL that comprises CDRs containing theamino acid sequences shown in SEQ ID NOs: 39, 41, and 43 (correspondingto CDR1, CDR2, and CDR3, respectively, in VH of P19-7D). A triabody anda tetrabody have a trimeric structure and a tetrameric structure,respectively, based on the single-stranded Fv structure, similarly to adiabody. A triabody and a tetrabody may be a trivalent antibody fragmentand a tetravalent antibody fragment, respectively, and may also bemulti-specific antibodies. Furthermore, the above term “a fragmentthereof” refers to an antibody fragment identified using a phage displaylibrary (e.g., see McCafferty et al., 1990, Nature Vol. 348: 552-554)and having antigen binding capacity. In addition, for example, see Kuby,J., Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York (1998).

The term “HCV infection” refers to a process during which HCV particlesbind to the cell surfaces of host cells, are proliferated in the hostcells, and then are released outside the cells. Therefore, the term“inhibiting (inhibition of) HCV infection” as used herein refers toinhibition or suppression of at least one of the above series of stepsof HCV infection. Preferably, a pathway for HCV to bind to virusreceptors on the surfaces of host cells and/or a pathway for an HCVgenome to enter the host cells is inhibited.

1-2. HCV Particles as Antigens

The anti-HCV antibody in the present invention is characterized byreacting with an HCV particle produced from a chimeric HCV genome as anantigen.

The term “HCV particles” refers to HCVs comprising HCV envelope proteinsand the HCV genome packaged therein.

The term “chimeric HCV genome” refers to an HCV genome derived from twoor more different HCV genomes. For example, the HCV genome is generallycomposed of RNA consisting of, from the 5′ side to the 3′ side, the 5′untranslated region, the core protein-coding sequence (hereinafter,referred to as “core sequence”), the E1 protein-coding sequence(hereinafter, referred to as “E1 sequence”), the E2 protein-codingsequence (hereinafter, referred to as “E2 sequence”), the p7protein-coding sequence (hereinafter, referred to as “p7 sequence”), theNS2 protein-coding sequence (hereinafter, referred to as “NS2sequence”), the NS3 protein-coding sequence (hereinafter, referred to as“NS3 sequence”), the NS4A protein-coding sequence (hereinafter, referredto as “NS4A sequence”), the NS4B protein-coding sequence (hereinafter,referred to as “NS4B sequence”), the NS5A protein-coding sequence(hereinafter, referred to as “NS5A sequence”), the NS5B protein-codingsequence (hereinafter, referred to as “NS5B sequence”), and the 3′untranslated region. Therefore, the “chimeric HCV genome” ischaracterized in that each of the above regions composing the HCV genomeis composed of regions derived from two or more different HCV strains.

Two or more different HCV strains composing each region in the abovechimeric HCV genome are not particularly limited. Examples thereofinclude the JFH-1 strain (genotype 2a), the J6CF strain (genotype 2a),and the TH strain (genotype 1b). Also, original HCV strains from whichregions constituting the chimeric genomes are derived and combinationsthereof are not particularly limited. This is specifically explainedbelow. Such a chimeric HCV genome comprises, for example, (i) the 5′untranslated region, the core sequence, the E1 sequence, the E2sequence, the p7 sequence, and the NS2 sequence, which may be derivedfrom strains other than the JFH-1 strain, and (ii) the NS3 sequence, theNS4A sequence, the NS4B sequence, the NS5A sequence, the NS5B sequence,and the 3′ untranslated region, which may be derived from the JFH-1strain. Alternatively, such a chimeric HCV genome comprises (i) the 5′untranslated region, the core sequence, the E1 sequence, the E2sequence, and the p7 sequence, which may be derived from strains otherthan the JFH-1 strain, and (ii) the NS2 sequence, the NS3 sequence, theNS4A sequence, the NS4B sequence, the NS5A sequence, the NS5B sequence,and the 3′ untranslated region, which may be derived from the JFH-1strain.

In one embodiment, such a chimeric HCV genome comprises, from the 5′side to 3′ side, (i) the 5′ untranslated region, the core sequence, theE1 sequence, the E2 sequence, and the p7 sequence, which are derivedfrom the J6CF strain, and (ii) the NS2 sequence, the NS3 sequence, theNS4A sequence, the NS4B sequence, the NS5A sequence, the NS5B sequence,and the 3′ untranslated region, which are derived from the JFH-1 strain.Preferably, such a chimeric HCV genome comprises (i) the 5′ untranslatedregion, the core region, the E1 region, the E2 region, the p7 region,and the sequence encoding 16 amino acid residues from the N-terminalside of the NS2 region, which are derived from the J6CF strain, and (ii)the sequence encoding 17^(th) amino acid residue from the N-terminalside and the following amino acid residues of the NS2 region, the NS3region, the NS4A region, the NS4B region, the NS5A region, the NS5Bregion, and the 3′ untranslated region, which are derived from the JFH-1strain. Such a chimeric genome has been cloned into J6/JFH-1 consistingof the nucleotide sequence of SEQ ID NO: 2. Furthermore, such a chimericgenome may comprise the 5′ untranslated region derived from the JFH-1strain, the core sequence, the E1 sequence, the E2 sequence, and the p7sequence, which are derived from the TH strain (Wakita, T. et al., J.Biol. Chem., 269, 14205-14210, 1994, JP Patent Publication (Kokai) No.2004-179), and the NS2 sequence, the NS3 sequence, the NS4A sequence,the NS4B sequence, the NS5A sequence, the NS5B sequence, and the 3′untranslated region, which are derived from the JFH-1 strain.Preferably, such a chimeric genome may comprise preferably the 5′untranslated region derived from the JFH-1 strain, the core sequence,the E1 sequence, the E2 sequence, the p7 protein, and the sequenceencoding 33 amino acid residues from the N-terminal side of the NS2region, which are derived from the TH strain, the sequence encoding the34^(th) amino acid residue from the N-terminal side and the followingamino acid residues of the NS2 region, the NS3 sequence, the NS4Asequence, the NS4B sequence, the NS5A sequence, the NS5B sequence, andthe 3′ untranslated region, which are derived from the JFH-1 strain.

Also, in one embodiment, HCV particles produced from the chimeric HCVgenome as described herein are inactivated by a method described later,so that the HCV particle can also be used as a vaccine. Inoculation ofthe vaccine to a human according to a method known in the art makes itpossible to directly produce the anti-HCV antibody of the presentinvention in vivo in the inoculated subject.

1-3. Preparation of HCV Particles

Infectious HCV particles to be used as antigens in the present inventioncan be prepared with a cell culture system. Basic techniques forpreparation of infectious HCV particles are described in WO04104198A1,WO06022422A1, WO06096459A2, Wakita, T. et al., Nat. Med. 11: 791-796,2005, Lindenbach, B D. et al., Science 309: 623-626, 2005, andPietschmann, T. et al., Proc. Natl. Acad. Sci. USA. 103:7408-7413, 2006.Preparation of infectious HCV particles is specifically described below.

1-3-1. Chimeric HCV Genome

A chimeric HCV genome as described in the above section, “1-2. HCVparticle as antigen,” can be used for preparation of HCV particles. Forexample, a nucleic acid having the nucleotide sequence shown in SEQ IDNO: 2 can be used. Also, a chimeric HCV genome to be used herein may beeither RNA or DNA. However, when a nucleic acid is RNA, “thymine (T)” inthe above nucleotide sequence is read as “uracil (U).” In thisdescription, the same applies to other nucleotide sequences.

1-3-2. Preparation of Chimeric HCV Genome RNA

HCV particles can be prepared by synthesizing chimeric HCV genome RNAfrom an expression vector constructed by ligating the cDNA offull-length chimeric HCV genome RNA downstream of a transcriptionpromoter so as to enable expression (e.g., a vector resulting frominsertion of a chimeric HCV genome under control of T7 promoter), andthen introducing the genomic RNA into host cells. Techniques known inthe art may be used for synthesizing such genomic RNA. For example, whenthe above expression vector is used, genomic RNA can be synthesized byan in vitro RNA synthesis method. Various kits for which such an invitro RNA synthesis method is employed are commercialized bymanufacturers relating to life science (e.g., MEGAscript T7 kit;Ambion). Genomic RNA may be synthesized using such a kit.

1-3-3. Host Cell

Examples of host cells, into which the thus synthesized chimeric HCVgenome RNA is introduced, are not particularly limited, as long as thesecells allow formation of HCV particles. Examples thereof includecultured cells such as Huh7, HepG2, IMY-N9, HeLa, HEK293, and the likemore preferably liver-derived cultured cells such as Huh7, and furtherpreferably derivative strains of Huh7, such as Huh7.5 and Huh7.5.1. Theterm “derivative strain” in the present invention refers to a cell linethat is induced from a cultured cell and differs from its originalstrain. Also, in Huh7, HepG2, IMY-N9, HeLa, or HEK293 cells, in whichthe CD81 gene and/or the Claudin1 gene is expressed, can also be used.

1-3-4. Method for Introducing Chimeric HCV Genome RNA into Host Cell

As a method for introducing chimeric HCV genome RNA into the above hostcells, any known method can be employed. Examples thereof includecalcium phosphate coprecipitation, a DEAE dextran method, lipofection,microinjection, and electroporation. Preferable examples thereof includelipofection and electroporation and an even more preferable examplethereof is electroporation.

The capacity of host cells, in which chimeric HCV genome RNA has beenintroduced, for viral particle production can be evaluated by atechnique known in the art. For example, such capacity can be confirmedby ELISA (Enzyme-Linked Immuno Sorbent Assay) method, western blotting,or the like with the use of an antibody reacting with a protein thatconstitutes HCV particles, such as the core protein, the E1 protein, orthe E2 protein released in the culture solution. Also, chimeric HCVgenome RNA contained in the HCV particles in the culture solution may beamplified via RT-PCR using specific primers for detection to indirectlydetect the presence of HCV particles.

1-3-5. Verification of Infectious Ability of HCV Particles

Whether or not the prepared HCV particles are infectious can beconfirmed by a method for evaluation of viral infection, which is knownin the art. For example, a supernatant obtained by culturing cells intowhich chimeric HCV genome RNA has been introduced is further added tonew HCV permissive cells (e.g., Huh7 cells), and then culturing cellsfor appropriate time period (e.g., 48 hours). Subsequently, the cellsare sufficiently washed, and then immunostained with an antibody thatrecognizes an HCV-specific protein such as anti-core antibody to countthe number of infected cells. Alternatively, evaluation can be carriedout by extracting a protein from the above cells, subjecting the extractto electrophoresis on SDS-polyacrylamide gel, and then detecting theHCV-specific protein via western blotting.

1-3-6. Purification of HCV Particles

The thus obtained HCV particles are preferably purified before use asantigens. HCV particles can be purified by removing cells and/or cellresidues from a solution (virus solution) containing infectious HCVparticles obtained by culturing the above cells into which the chimericHCV genome RNA has been introduced, carrying out column chromatographyor density-gradient centrifugation or a combination of the two in anyorder. Cells and residues thereof can be removed by centrifugationand/or filtration, for example. If necessary, a virus solution fromwhich residues have been removed can be concentrated approximately 10-to 100-fold using an ultrafiltration membrane with a molecular weightcut off ranging from 100,000 to 500,000.

Examples of the above column chromatography include gel filtrationchromatography, ion exchange chromatography, and affinitychromatography. A preferable example of gel filtration chromatography ischromatography using a cross-linked polymer of allyl dextran andN,N′-methylenebisacrylamide as the gel matrix. A more preferable examplethereof is chromatography comprising Sephacryl (registered trademark)S-300, S-400, and S-500. An example of ion-exchange chromatography is amethod using Q-Sepharose (registered trademark) as an anion exchangeresin and SP Sepharose (registered trademark) as a cation exchangeresin. For affinity chromatography, a resin binding heparin, sulfatedcellulofine, lectin, and a substrate selected from various dyes boundthereto as a ligand can be used as a support. A preferable examplethereof is affinity chromatography using HiTrap Heparin HP (registeredtrademark), HiTrap Blue HP (registered trademark), HiTrap Benzamidine FF(registered trademark), and sulfated Cellulofine, and a support to whichLCA, ConA, RCA-120, and WGA have been bound. More preferably, HCVparticles are purified by affinity chromatography using sulfatedCellulofine as a support. With the use of this method, HCV particles canbe purified 30-fold or more in terms of the ratio of the HCV RNA copynumber to the total protein amount in the solution.

As a solute for the formation of density gradient in density-gradientcentrifugation, cesium chloride, sucrose, Nycodenz (registeredtrademark), and a sugar polymer such as Ficoll (registered trademark)and Percoll (registered trademark) can be used. Sucrose can be furtherpreferably used. As a solvent, preferably, water or a buffer, such asphosphate, Tris, acetate, or glycine buffer, can be used to be used fordensity-gradient centrifugation. The centrifugal force that is employedupon purification via density-gradient centrifugation preferably rangesfrom 1×10⁴ g to 1×10⁹ g, more preferably ranges from 5×10⁴ g to 1×10⁷ g,and most preferably ranges from 5×10⁴ g to 5×10⁵ g.

The temperature for purification of the above HCV particles preferablyranges from 0° C. to 40° C., more preferably ranges from 0° C. to 25°C., and most preferably ranges from 0° C. to 10° C.

When purification is carried out via density-gradient centrifugation incombination with column chromatography, preferably, HCV particles arefirst purified through a plurality of chromatography columns followed bydensity-gradient centrifugation. More preferably, a fraction containingHCV particles obtained via anion exchange column chromatography followedby affinity chromatography is subjected to purification viadensity-gradient centrifugation. Most preferably, a fraction containingHCV particles obtained with the use of a Q-Sepharose (registeredtrademark) column is further purified with the use of a sulfatedcellulofine-based column, and the resulting fraction containing HCVparticles is then purified via density-gradient centrifugation. Duringor after the steps of column chromatography and density-gradientcentrifugation, dialysis or ultrafiltration may be carried out tosubstitute a solute of a solution containing HCV particles and/or toconcentrate HCV particles.

1-3-7. Inactivation of HCV Particles

Inactivation of HCV particles to be used as antigens is not essentialfor preparation of the anti-HCV antibody of the present invention, whenHCV particles are administered to non-human animals. However,inactivation is preferably carried out in terms of prevention ofinfection of a surgeon. Also, when an HCV particle produced from theabove chimeric HCV genome is administered as a vaccine to a human,inactivation is essential. HCV particles can be inactivated by adding aninactivator such as formalin, β-propiolactone, or glutardialdehyde to avirus solution containing infectious HCV particles, and thensufficiently mixing the resultant (Appaiahgari et al., Vaccine, 22:3669-3675, 2004). Further, a virus solution containing infectious HCVparticles can be irradiated with ultraviolet rays to rapidly eliminateinfectivity of HCV. Irradiation with ultraviolet rays is preferred sinceit realizes virus inactivation with little influence on proteins or thelike that constitute HCV. A source of ultraviolet rays used forinactivation can be a commercially available germicidal lamp. Inparticular, a 15 w germicidal lamp can be used, although the source isnot limited thereto. Preferably, infectious HCV particles can beinactivated by irradiating a solution containing infectious HCVparticles with ultraviolet rays at 20 mW/cm² at room temperature for atleast 5 minutes. Moreover, such an inactivation method is not limited bythe purified or unpurified state.

1-4. Preparation of Anti-HCV Antibody 1-4-1. Immunization of Animal

The antibody of the present invention can be obtained by administeringHCV particles to animals to induce the antibody via immune reaction.Examples of animals to be used for immunization are not particularlylimited, as long as they are non-human animals capable of producingantibody-producing cells with which hybridomas can be prepared. Forexample, non-human mammals and more specifically mice, rats, hamsters,guinea pigs, rabbits, goats, donkeys, sheep, camels, and horses can beused. In the present invention, examples involving the use of mice aredescribed below.

Four- to 10-week-old mice may be immunized with the HCV particle antigen(immunogen) obtained in the above method. According to circumstances,the step of purification of HCV particles may be altered or omitted, andHCV particle inactivation may be omitted.

An HCV particle as an immunogen is dissolved in a buffer, so as toprepare an immunogen solution. At this time, an adjuvant may be added ifnecessary for effective immunization. Examples of an adjuvant includeFreund's complete adjuvant (hereinafter, referred to as “FCA”), Freund'sincomplete adjuvant (hereinafter, referred to as “FIA”), aluminiumhydroxide gel, Hemophilus pertussis vaccine, Titer Max Gold (Vaxel),GERBU adjuvant (GERBU Biotechnik), and MPL (Monophosphoryl Lipid A)+TDM(synthetic trehalose dicorynomycolate) (Sigma Adjuvant System; Sigma).These adjuvants may be used independently or mixed and then used.

Next, an immunogen solution prepared as described above is administeredto a mouse (e.g., the inbred mouse strain Balb/c) for immunization.Examples of a method for administration of such an immunogen solutioninclude, but are not limited to, subcutaneous injection of FIA or FCA,intraperitoneal injection using FIA, and intravenous injection of 0.15mol/L sodium chloride. A single dose of the immunogen is arbitrarilydetermined depending on, for example, the type of an animal to beimmunized or the route of administration, to be generally about 50 μg to200 μg per animal. Also, the intervals of immunization are notparticularly limited (e.g., intervals of several days to several weeks,and preferably intervals of 1 to 4 weeks). After the primaryimmunization, boost immunization is preferably carried out 2 to 6 times,and preferably 3 or 4 times. Blood is collected from the venous plexusof the ocular fundus or caudal vein of the immunized mouse after theinitial immunization and the antibody titer in the serum is preferablymeasured by ELISA or the like. Also, the activity of inhibiting HCVinfection can also be measured. When the antibody titer reaches aplateau, the immunogen solution is injected intravenously orintraperitoneally to complete the final immunization. Preferably, noadjuvant is used for the final immunization. On days 3 to 10 after thefinal immunization, preferably on day 4 after the final immunization,blood is collected from the immunized mouse, serum is treated accordingto a known method (Antibodies: A Laboratory Manual Cold Spring HarborLaboratory, 1988), and thus a polyclonal antibody can be obtained. Inaddition, whether or not an immunized animal produces the anti-HCVantibody of the present invention capable of inhibiting HCV infectioncan be preferably confirmed in advance, specifically, before the finalimmunization, by the methods according to the above-mentioned “1-3-5.Verification of infectious ability of HCV” and “1-4-6. Selection ofanti-HCV antibody (described later).”

1-4-2. Preparation of Hybridoma Cell Producing Anti-HCV MonoclonalAntibody

When an anti-HCV monoclonal antibody is prepared, a hybridoma producingthe antibody can be prepared by a method described below, for example.

First, antibody-producing cells are collected from the above-immunizedmouse. Examples of antibody-producing cells include spleen cells, lymphnode cells, and peripheral blood cells. Spleen cells or local lymph nodecells are preferable. Subsequently, cell fusion of antibody-producingcells with myeloma cells is performed, so that a hybridoma producing ananti-HCV monoclonal antibody can be prepared. Myeloma cells to be usedfor cell fusion are not particularly limited, as long as they aremouse-derived established cells and capable of growing in vitro. Forconvenient selection of a hybridoma in a step described later,preferable myeloma cells have drug selectivity, so that they cannotsurvive in an unfused state in selective medium (e.g., HAT medium; thatis, Dulbecco's modified MEM (hereinafter, referred to as “DMEM”)supplemented with 5×10⁻⁵M 2-mercaptoethanol, 100 units/mL penicillin,100 μg/mL streptomycin, and 10% fetal calf serum (hereinafter, referredto as “FCS”), 10⁻⁴ M hypoxanthine, 1.5×10⁻⁵ M thymidine, and 4×10⁻⁷ Maminopterin), but they can survive only in a state fused toantibody-producing cells. For example, 8-azaguanine-resistant mouse(BALB/c-derived) myeloma cell lines P3-X63Ag8-U1 (P3-U1), SP2/0-Ag14(SP2/0), P3-X63-Ag8653 (653), P3-X63-Ag8 (X63), and P3/NS1/1-Ag4-1 (NS1)can be used. These cell lines are available from RIKEN BioResourceCenter, ATCC (American Type Culture Collection), or ECACC (EuropeanCollection of Cell Cultures). Culture and subculture are carried out inaccordance with a conventional technique (e.g., Antibodies: A LaboratoryManual Cold Spring Harbor Laboratory, 1988, Selected Methods in CellularImmunology W.H. Freeman and Company, 1980).

For the above cell fusion of antibody-producing cells and myeloma cells,spleen cells and myeloma cells obtained as described above are washed,antibody-producing cells and myeloma cells are mixed at a ratio rangingfrom about 1:1 to 20:1 in medium for culturing animal cells, such asserum-free DMEM or RPMI1640 medium, and then fusion reaction isconducted in the presence of a cell fusion accelerator. As a cell fusionaccelerator, polyethylene glycol (hereinafter, referred to as “PEG”)having an average molecular weight ranging from 1,500 to 4,000 Da can beused at a concentration ranging from about 10% to 80%, for example. Ingeneral, PEG with an average molecular weight of 1,500 Da is preferablyused. An auxiliary agent such as dimethyl sulfoxide can be used incombination to enhance the fusion efficiency, if necessary. Furthermore,antibody-producing cells can be fused to myeloma cells using acommercially available cell fusion apparatus utilizing electric stimuli(e.g., electroporation) (Nature, 1977, Vol. 266, 550-552).

After cell fusion treatment, cells are washed with medium used forculturing myeloma cells (e.g., DMEM supplemented with 5×10⁻⁵ M2-mercaptoethanol, 100 units/mL penicillin, 100 μg/mL streptomycin, and10% FCS). A cell suspension is prepared, appropriately diluted withFCS-containing RPMI1640 medium or the like, for example, and then addedonto a 96-well plate at about 2×10⁶ cells/well. Selective medium isadded to each well, and then cells are cultured continuously whileappropriately exchanging selective media. The culture temperature rangesfrom 20° C. to 40° C. and is preferably about 37° C. When the myelomacells are of an HGPRT-deficient cell line or thymidine kinase(TK)-deficient cell line, only the hybridomas of antibody-producingcells and myeloma cells can selectively be cultured and grown in theselective medium containing hypoxanthine, aminopterin, and thymidine(HAT medium). As a result, cells that start to grow on about day 10after the initiation of culture in the selective medium can be selectedas hybridoma cells.

1-4-3. Test for Activity of Inhibiting HCV Infection

The presence or the absence of the activity of inhibiting HCV infectionof antibodies produced by the above hybridomas can be determined bymethods illustrated below and methods using infectious HCV particles,which described later in Examples. For example, first, the aboveanti-HCV monoclonal antibody-producing hybridoma is cultured, and then aportion of the culture supernatant is collected as an antibody sample.The antibody sample is mixed with the above infectious HCV particles,and then the reaction is conducted at 37° C. for 1 hour (mixed sample).Next, 50 μL of the mixed sample is added to the Huh7 cells cultured onthe previous day at 5×10³ cells/well on a 96-well plate, followed by 2.5hours of culture at 37° C. After culture, the culture solution and themixed sample are removed, cells are washed with PBS, a fresh medium isadded again, and culture is continued. The culture supernatant isremoved 48 hours later, cells are washed once with PBS, 100 μL of ISOGEN(Nippon Gene) is added to prepare RNA from the cells, RNA is quantified,and the amount of HCV genomic RNA is then measured. HCV RNA is detectedvia quantitative RT-PCR by detecting RNA in the 5′ untranslated regionof HCV RNA according to the method of Takeuchi et al. (Gastroenterology,116: 636-642, 1999).

Alternatively, the activity of inhibiting HCV infection can be evaluatedby the following method. First, the antibody sample is mixed withinfectious HCV particles obtained from an anti-HCV monoclonalantibody-producing hybridoma cell line, and the mixture is subjected toa reaction at 37° C. for 1 hour (mixed sample). Subsequently, 50 μL ofthe mixed sample is added to the Huh7 cells cultured on the previous dayat 1×10⁴ cells/well on a 96-well plate, and then cells are cultured at37° C. for 2.5 hours. After culture, the culture solution and the mixedsample are removed, cells are washed with PBS, a fresh medium is addedagain, and culture is continued. The culture solution is removed 72hours later, the plate is introduced into ice-cold methanol to fixcells. Thereafter, methanol is removed via air drying, and cells arepermeabilized with the use of Block Ace (registered trademark)(Dainippon Pharmaceutical Co., Ltd.) containing 0.3% Triton (registeredtrademark)-X 100 (GE Healthcare). The number of HCV-infected cells iscounted under a fluorescent microscope using a clone 2H9 anti-HCV-coreantibody (see Nat Med., 2005, 11: pp. 791-6) and goat anti-mouseIgG-Alexa488 (Molecular Probes), and the antibody samples in the wellsin which HCV infection is inhibited can be selected as an anti-HCVmonoclonal antibody having activity of inhibiting HCV infection.

Alternatively, a test can be conducted for the activity of inhibitingHCV infection with the use of infectious HCV-like particles(hereinafter, referred to as “HCVpp”) that are prepared by causing thedisplay of functional HCV envelope proteins on retrovirus particlesinstead of infectious HCV particles. A green fluorescent protein (GFP)marker gene or a luciferase gene is packaged within HCVpp, making itpossible to rapidly measure with high reliability infection mediated byHCV envelope proteins (Bartosch, B. et al. J. Exp. Med. 197: 633-642,2003). Specifically, HCVpp having envelope proteins of genotype 2a canbe obtained as follows, for example. A pcDNA J6dC-E2 vector isconstructed by cloning a nucleic acid that encodes the 132″ to the750^(th) amino acid residues (corresponding to a part of the coreprotein, the E1 protein, and the E2 protein) of the protein (NCBIProtein Accession No. AAF01178.1) of the J6CF strain (an HCV strain ofgenotype 2a) into pcDNA3.1. A Gag-Pol 5349 expression vector isconstructed by cloning genes encoding gag and pol of a mouse leukemiavirus into the vector. A Luc126 retrovirus vector is constructed bycloning a luciferase gene into the vector. The vectors are transfectedinto HEK293T cells (ATCC CRL-1573) using FuGENE6 (Roche: catalog No.11814443001). After transfection, a culture solution containing HCVpp iscollected and then filtered with a 0.45-μm membrane filter, so thatHCVpp having envelope proteins of genotype 2a can be obtained.

For preparation of HCVpp having envelope proteins of genotype 1a, apcDNA H77dC-E2 vector can be used instead of the above pcDNA J6dC-E. ThepcDNA H77dC-E2 vector is constructed by cloning a nucleic acid thatencodes the 132″ to the 746^(th) amino acid residues (corresponding to apart of the core protein, the E1 protein, and the E2 protein) of aprotein (NCBI Protein accession No. AAB67036.1) of the H77 strain, whichis an HCV strain of genotype 1a, into pcDNA3.1.

For preparation of HCVpp having envelope proteins of genotype 1b, apcDNA THdC-E2 vector can be used instead of the above pcDNA J6dC-E. ThepcDNA THdC-E2 vector is constructed by cloning a nucleic acid thatencodes the 132″ to the 747^(th) amino acid residues (corresponding to apart of the core protein, the E1 protein, and the E2 protein) of aprotein of the TH strain, which is an HCV strain of genotype 1b,(Wakita, T. et al., J. Biol. Chem., 269, 14205-14210, 1994) intopcDNA3.1.

For example, HCVpp is mixed with the antibody sample obtained from ananti-HCV monoclonal antibody-producing hybridoma cell line, and then themixture is allowed to react at 37° C. for about 30 minutes. The antibodysample is diluted with DMEM (DMEM containing 10% FCS, 1% MEMnonessential amino acid solution, 10 mM HEPES-Tris (pH 7.3), and 1 mMsodium pyruvate). The above mixture of HCVpp and the antibody sample isadded to Huh7.5.1 cells cultured for 1 day on a 96-well plate at 1×10⁴cells/well, followed by about 3 hours of culture at 37° C. Afterculture, the sample is removed, cells are washed once with PBS, freshmedium is added, and then culture is continued. After about 72 hours,the culture solution is removed. Cells are washed about 4 times withPBS, 25 μL/well serum-free DMEM and 25 μL/well lysis buffer (e.g.,Steady-Glo (Promega: catalog No. E2520) can be used) are added, and thencells are lysed. (When a kit is used, cell lysis is basically performedaccording to instructions included therewith.) The cell lysis solution(40 μL/well) is transferred to a white 96-well plate (e.g., SumitomoBakelite Co., Ltd.: catalog No. MS-8496W), and then luminescenceintensity is measured using an apparatus with which fluorescence can bemeasured (e.g., ARVO X4 (PerkinElmer)). Luminescence intensity (%)obtained after mixing with DMEM is regarded as representing 100%infection. Thus infection (%) after mixing with an antibody sample canbe found. Therefore, an antibody sample that results in a decrease ininfection (%) (that is, when the addition thereof results in lowluminescence intensity) can be determined to be an anti-HCV monoclonalantibody having activity of inhibiting HCV infection.

In addition, the antibody-producing hybridoma of the present inventionis not particularly limited, as long as it is a hybridoma that isselected by the above method. A specific example of such a hybridoma isa hybridoma cell line producing “P18-9E” or “P19-7D” monoclonal antibodyhaving activity of inhibiting HCV infection of the present invention(described later).

1-4-4. Preparation of Anti-HCV Antibody

The hybridomas selected above are conditioned to serum-free medium, suchas Hybridoma-SFM (Invitrogen), and the cultured supernatant can bedesignated as an anti-HCV antibody sample (anti-HCV monoclonal antibodysample). Culture can be conducted with the use of a flask, petri dish,spinner culture bottle, roller bottle, or high-density culture flask(CELLine, Becton, Dickinson and Company).

When monoclonal antibodies are prepared from animals, the anti-HCVmonoclonal antibody-producing hybridoma cells selected above areintraperitoneally injected into pristane-treated 8- to 10-week-old mice,nude mice, or SCID mice (0.5 mL of 2,6,10,14-tetramethylpentadecane(pristane) is administered intraperitoneally and mice are grown for 2weeks) at 2×10⁷ to 5×10⁶ cells/mouse. Hybridomas experience ascitestumor formation within 10 to 21 days. Ascites fluid is sampled from themice or the like to prepare anti-HCV monoclonal antibody samples. Theobtained antibody samples are centrifuged to remove cells or disruptedcells, the samples are subjected to salting-out with 40% to 50%saturated ammonium sulfate, caprylic acid precipitation, DEAE-Sepharosecolumn, Protein-A column, Protein-G column, HiTrap IgM PurificationHP-column (GE Healthcare), mannan binding protein-column (Pierce), orgel filtration column, and such techniques are carried out alone or inadequate combination to recover IgG or IgM fractions. Thus, purifiedanti-HCV monoclonal antibodies can be obtained. The purified monoclonalantibody subclasses are determined with the use of, for example a mousemonoclonal antibody typing kit (Pierce). The classes of the antibodieshaving activity of inhibiting HCV infection of the present invention arenot particularly limited. Such an antibody class is preferably IgG orIgM and IgM antibodies are more preferable.

1-4-5. Preparation of Anti-HCV Humanized Antibody

For anti-HCV humanized antibodies, FRs of human antibodies with whichCDRs can form good antigen-binding sites are selected. According toneed, amino acids in FRs in the antibody V regions may be substituted,so that the complementarity determining regions (CDRs) of the reshapedhuman antibodies form adequate antigen-binding sites (Sato, K., et al.,Cancer Res. 53: 851-856, 1993).

When humanized anti-HCV antibodies of the present invention areprepared, for example, mRNA is extracted from anti-HCV monoclonalantibody-producing hybridomas, so as to synthesize cDNA encoding VH andVL. The thus synthesized cDNA is inserted into a phage or plasmid vectorto construct a cDNA library. Recombinant phages or plasmids having cDNAencoding VH and recombinant phages or plasmids having cDNA encoding VLare separately isolated from the resulting library with the use of the Cor V region of the mouse antibody as a probe. The whole nucleotidesequences of VH and VL of the target antibody on the recombinant phageor plasmid are determined, and the whole amino acid sequences of VH andVL are deduced based on the nucleotide sequences.

Alternatively, cDNA encoding VH and VL can be cloned via PCR. cDNAs ofhybridomas prepared in the above-described manner are used as templates,the templates are amplified using a plurality of primers designed basedon the amino acid sequences conserved in the relevant genes, and thecDNA fragments are cloned into cloning vectors. Thus, cDNA encoding VHand VL can be obtained. cDNA encoding VH and VL of the anti-HCV antibodymay be inserted into a region upstream of the gene encoding CH and CL ofthe human antibody to construct cDNA encoding a human anti-HCVmonoclonal antibody. In CH, Cγ1, Cγ2, Cγ3, and Cγ4 can be used, and inCL, Cκ and Cλ can be used. In order to improve stability of the antibodyor production thereof, the C region may be modified.

When mammalian cells are used as host cells, the gene of interest can beexpressed using an expression vector containing a promoter that canexpress the gene of interest in mammalian cells, the antibody gene to beexpressed, and poly A signal operably linked to a site downstream of the3′ end. Examples of promoters that can be used herein include viruspromoters/enhancers, such as human cytomegalovirus, retrovirus, polyomavirus, adenovirus, and simian virus 40 (SV40) and promoters/enhancersderived from mammalian cells, such as the elongation factor 1α (EF1α).

When E. coli is used as a host, the gene of interest can be expressedwith the use of an expression vector in which a promoter that canexpress the gene of interest in E. coli cells, a signal sequence forantibody secretion, and the antibody gene to be expressed are operablylinked. Examples of promoters include lacZ promoter, araB promoter, Trppromoter, and T7 promoter.

When insect cells are used as host cells, the gene of interest can beexpressed with the use of an expression vector containing a promoterthat can express the gene of interest in insect cells, the antibody geneto be expressed, and poly A signal operably linked to a site downstreamof the 3′ end. Examples of promoters that can be used herein includepolyhedrin promoter and baculovirus OpNMPV-derived immediate-early OpIE2promoter.

Host cells containing the above expression vector for expression of ananti-HCV humanized antibody of the present invention are cultured underappropriate conditions according to a conventional technique, so thatthe antibody is produced in the culture solution supernatant or withinthe host cells. Specifically, for example, when cultured cells of amammal are used as host cells, the host cells are seeded in DMEM at1×10⁵ cells/mL, and then cultured with a 5% CO₂ incubator at 37° C., sothat a culture solution supernatant containing the antibody can beobtained. Also, for example, when Escherichia coli is used as a hostcell, the host cells are seeded and cultured in medium such as LBmedium, which is generally used for culturing Escherichia coli, so as toinduce protein expression. Thus, the antibody of interest can beproduced in the culture solution supernatant or within the host cells.

In addition, an anti-HCV humanized antibody of interest can be purifiedand recovered from a culture solution supernatant or a disrupted cellsolution through selection for the C region using a protein A column, aprotein G column, an anti-immunoglobulin antibody affinity column, orthe like.

1-4-6. Selection of Anti-HCV Antibody

For selection of the anti-HCV antibody of the present invention, HCVproteins are immobilized (i.e., solid-phased) on a support, the antibodysample is added, and then reaction is allowed to proceed for a period oftime under conditions sufficient for the formation of anantibody/antigen complex. Next, for detection of the thus formedcomplex, a secondary antibody; that is, an antibody that has an enzyme,dye, or radioisotope as a signal and that recognizes the antibody sampleis brought into contact with the resultant, so as to form a secondmixture. The second mixture is allowed to react for a period of timeunder conditions sufficient for the formation of the antibody/antigencomplex. Thus, the presence of the antibody that recognizes the HCVprotein is detected with the aid of the signal of the enzyme, dye, orradioisotope.

As HCV proteins to be immobilized on a support, HCV particles may beused. Alternatively, proteins expressed in E. coli, yeast, mammaliancells, insect cells, or the like with the use of cDNA consisting of thecore sequence, the E1 sequence, the E2 sequence, the p7 sequence, theNS2 sequence, the NS3 sequence, the NS4A sequence, the NS4B sequence,the NS5A sequence, or the NS5B sequence of the HCV genome may be used.Further, such proteins may be chemically synthesized and used. Thelength of the amino acid residues composing a protein to be used forimmobilization is not limited, and such length is 3 or more amino acidresidues and more preferably 8 or more amino acid residues.

Expression of the E1 protein and the E2 protein that are envelopeproteins of the JFH-1 strain or the J6CF strain in mammalian cells is asdescribed below, for example. The E1 protein of the JFH-1 strain or theJ6CF strain (JFH1 strain: NCBI Protein Accession No. BAB32872, J6CFstrain: NCBI Protein Accession No. AAF01178.1) starts from the 192″amino acid residue and ends at the 383^(rd) amino acid residue, when theinitiator methionine of the full-length amino acid sequence of theprotein of the JFH-1 strain is regarded as the 1^(st) amino acidresidue. The portion ranging from the 353^(rd) to the 383^(rd) aminoacid residues of the E1 protein is considered to be a transmembranedomain (also referred to as “C-terminal hydrophobic domain”) (Cocquerel,L. et al., J. Virol. 74: 3623-3633, 2000).

The E2 protein of the JFH-1 strain or the J6CF strain starts from the384^(th) amino acid residue and ends at the 750^(th) amino acid residueof the above amino acid sequence. The portion ranging from the 722″ tothe 750^(th) amino acid residues of the E2 protein is considered to be atransmembrane domain (Cocquerel, L. et al., J. Virol. 74: 3623-3633,2000).

When proteins are secreted after the expression in the culturesupernatant of mammalian cells, it is necessary for such proteins tohave signal peptides, but it is not necessary for them to havetransmembrane domains.

Accordingly, the E1 or E2 protein containing no transmembrane domaincomprises the above 192″ to the 352″ amino acid residues and the above384^(th) to the 721^(st) amino acid residues, respectively. Shift of anamino acid location is not problematic, provided that the amino acidsare qualitatively equivalent to each other. A sequence ranging from the192^(nd) to the 352^(nd) amino acid residues of the E1 protein of theJFH-1 strain, and a sequence ranging from the 384^(th) to the 720^(th)amino acid residues (and preferably the sequence ranging from the384^(th) to the 714^(th) amino acid residues) of the E2 protein of theJFH-1 strain can be used as proteins that do not contain anytransmembrane domain. Also, a sequence ranging from the 192^(nd) to the352^(nd) amino acid residues of the E1 protein of the J6CF strain, and asequence ranging from the 384^(th) to the 720^(th) amino acid residuesof the E2 protein of the J6CF strain can be used as proteins that do notcomprise a transmembrane domain. When such amino acid residue numbersare applied to amino acid sequences of other HCV strains, sequences maybe designated with the amino acid residue numbers corresponding inalignment with the amino acid sequence of the relevant full-length JFH-1protein.

A nucleic acid that encodes a protein not containing any transmembranedomain of the E1 or E2 protein can be synthesized via PCR using cDNA ofthe JFH-1 strain as a template based on the nucleic acid sequence (NCBINucleotide Accession No. AB047639) of the JFH-1 strain given in GenBank,or such nucleic acid can be fully synthesized.

The corresponding E1 and E2 protein regions of an HCV strain other thanthe JFH-1 strain can be easily determined by aligning the sequences, sothat the lengths of matched portions (of the sequences) become thelongest when compared with the sequence of the JFH-1 strain, whiletaking substitution or deletion of the sequence of the HCV strain intoconsideration. Such analysis can be carried out using geneticinformation processing software (e.g., GENETYX, Software DevelopmentCo., Ltd.).

When the E1 or E2 protein containing no transmembrane domain is secretedafter expression in a mammalian cell, a nucleic acid encoding theprotein is ligated to a site downstream of a nucleic acid that encodes asignal peptide in such a manner that reading frames of codons arecorrectly aligned (i.e., in-frame), a stop codon is added to the 3′ end,and the resultant is then inserted into an expression vector. A signalpeptide is mainly composed of hydrophobic amino acids comprising 15 to30 amino acid residues located at the N terminus of the secretoryprotein and is involved in the mechanisms of protein transport throughthe cell membrane.

A signal peptide that can be used for protein secretion after expressionin a mammalian cell may be a signal peptide of a secretory protein.Examples of vectors having signal peptides include a vector having asignal peptide sequence of mouse GM-CSF (JP Patent Publication (kokai)No. 63-276490 A (1988)), a pSecTag/FRT/V5-His vector (Invitrogen) havinga signal peptide sequence of the IgG κ strand, a p3xFLAG-CMV13 vector(Sigma) having a signal peptide sequence of preprotrypsin, a pFUSE-Fc2vector (InvivoGen) having a signal peptide sequence of IL-2, and apTriEx-7 vector (Novagen) having a signal peptide sequence of IgM.

When a protein is expressed, such a protein is expressed as a fusionprotein of a target protein and a label protein, and the fusion proteincan be detected or purified with the use of an antibody reacting withthe label protein or a molecule that specifically binds thereto. Suchlabel proteins are also referred to as “tags.” Label proteins are notlimited, and examples thereof include FLAG peptide (also referred to asflag peptide or Flag peptide), 3×FLAG peptide (also referred to as3×FLAG peptide, 3× Flag peptide, or 3× flag peptide), HA peptide, 3×HApeptide, myc peptide, 6×His peptide, GST polypeptide, MBP polypeptide,PDZ domain polypeptide, alkaline phosphatase, immunoglobulin, andavidin. Such peptides or polypeptides are generally fused to the N- orC-terminus of the target proteins, but such peptides or polypeptides canbe inserted into the target proteins according to need. A vector havinga fusion polypeptide of preprotrypsin signal peptide and 3×FLAG peptideis available as the p3xFLAG-CMV-9 vector from Sigma.

1-4-7. Analysis of Antibody Epitope

A preferred example of the antibody having activity of inhibiting HCVinfection according to the present invention can recognize as an epitopethe conformation of the complex of the E1 protein and the E2 protein ofHCV, and of preferably the J6CF strain. Specifically, a preferredexample of the antibody according to the present invention can recognizeas an epitope the whole or a portion of the tertiary structure of thecomplex of the E1 protein and the E2 protein. The E1 protein and the E2protein are envelope proteins involved in binding with receptors (HCVreceptors) on the surfaces of host cells. HCV infects host cells via theHCV receptors. CD81 is identified as one of the HCV receptors, which hasbeen reported to be one of essential factors for HCV infection (Akazawaet al., J. Virol. 81: 5036-5045, 2007). Therefore, HCV maintaining itsability to infect cells is thought to retain the conformation of thecomplex (of the E1 protein and the E2 protein) capable of binding toCD81.

The anti-HCV antibody of the present invention recognizes as an epitopethe whole or a portion of the conformation of the complex of the E1protein and the E2 protein in HCV, and binds thereto. Binding of theanti-HCV antibody of the present invention to the complex of the E1protein and the E2 protein inhibits the binding of the E1 protein andthe E2 protein of infectious HCV to CD81 on host cells. As a result, HCVis thought to lose its infectious ability. Therefore, the anti-HCVantibody of the present invention is capable of binding to variousinfectious HCVs and thus is capable of inhibiting HCV infection.

HCV is known to tend to undergo mutation. A diversity-carrying HCVaggregate that proliferates while undergoing mutation is referred to as“quasispecies.” A hypervariable region (hereinafter, referred to as“HVR”) that frequently undergo amino acid mutation is located at theN-terminus of the E2 protein of HCV. It has been demonstrated that knownantibodies having activity of inhibiting HCV infection recognize apeptide of such a region that often undergoes mutation as an epitope(Farci et al., Proc. Natl. Acad. Sci. USA. 93: 15394-15399, 1996;Shimizu et al., J. Virol. 68: 1494-1500, 1994; Zhang et al., Proc. Natl.Acad. Sci. USA. 104: 8449-8454, 2007). However, it has been reportedthat an antibody that recognizes HVR as an epitope loses the effects dueto HCV mutation (Farci et al., Proc. Natl. Acad. Sci. USA. 91:7792-7796, 1994; Weiner et al., Proc. Natl. Acad. Sci. USA. 89:3468-3472, 1992; Kato et al., J. Virol. 67: 3923-3930, 1993). Therefore,an epitope for the anti-HCV antibody is preferably an epitope other thanHVR.

The anti-HCV antibody of the present invention recognizes as an epitopethe conformation of the complex of the E1 protein and the E2 protein.Since the anti-HCV antibody of the present invention differs from aconventional anti-HCV antibody having activity of inhibiting HCVinfection, which recognizes only HVR as an epitope, the anti-HCVantibody of the present invention can be expected to keep its effects ofinhibiting infection due to quasispecies of HCV. For analysis of anepitope of the antibody of the present invention produced by a hybridomacell line selected by the above method, enzyme immunoassay (EIA),western blotting, dot blotting, or the like using HCV proteins can beemployed. Through analysis of such an epitope as primary screening foran anti-HCV monoclonal antibody, an anti-HCV monoclonal antibodytargeting a specific HCV protein can be efficiently screened for.

2. Inhibitory Agent for HCV Infection

Another embodiment of the present invention is an inhibitory agent forHCV infection.

The term “inhibitory agent for HCV infection” in the present inventionrefers to a substance comprising the above anti-HCV antibody of thepresent invention as an active ingredient and being capable ofinhibiting at least one function required for the process of HCV toinfect host cells. The inhibitory agent for HCV infection of the presentinvention can be used independently as a medicament or an activeingredient of a pharmaceutical composition, or preferably used as amedicament for treating or preventing hepatitis C. Furthermore, theinhibitory agent can also be used as a research tool for elucidation ofthe HCV infection mechanism.

2-1. Pharmaceutical Composition

The inhibitory agent for HCV infection of the present invention can beused for a pharmaceutical composition. The pharmaceutical composition ofthe present invention can comprise pharmaceutically acceptable carriersin addition to the anti-HCV antibody of the present invention as amedicament.

The term “pharmaceutically acceptable carriers” refers to a solventand/or an additive that can be generally used in the technical field offormulation.

Examples of such pharmaceutically acceptable solvents include water andpharmaceutically acceptable organic solvents (e.g., ethanol, propyleneglycol, ethoxy-isostearyl alcohol, polyoxy-isostearyl alcohol, andpolyoxyethylene sorbitan fatty acid esters). These solvents aredesirably sterilized and preferably adjusted to be isotonic to blood asnecessary.

Also, examples of pharmaceutically acceptable additives includecollagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinylpolymer, carboxymethylcellulose sodium, sodium polyacrylate, sodiumalginate, water-soluble dextran, carboxymethyl starch sodium, pectin,methylcellulose, ethylcellulose, xanthan gum, gum Arabic, casein, agar,polyethylene glycol, diglycerine, glycerine, propylene glycol, vaseline,paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA),mannitol, sorbitol, lactose, and a surfactant, which is acceptable as apharmaceutical additive.

The pharmaceutical composition of the present invention can furthercontain, an excipient, a binder, a disintegrator, a filler, anemulsifier, a fluid agent for addition and regulation, a lubricant, ataste and flavor corrigent, a solubilizing agent (solubilizer), asuspension, a diluent, a surfactant, a stabilizer, an absorptionpromotor, an extending agent, a wetting agent, a moisturizing agent(e.g., glycerin and starch), an adsorbent, a disintegration-suppressingagent, a coating agent, a colorant, a preservative, an antioxidant,aroma chemicals, a flavor agent, a sweetening agent, and a bufferingagent, as necessary.

The above solvents and/or additives can be used independently or inappropriate combinations according to the dosage form of thepharmaceutical composition of the present invention to be used herein.For example, when the pharmaceutical composition of the presentinvention is used as a preparation for injection, a purified anti-HCVantibody is dissolved in a solvent (e.g., saline, buffer, and dextrosesolution), an adsorption-preventing agent (e.g., Tween80, Tween20,gelatin, and human serum albumin) is added to the solution, and thus theresultant can be used. Alternatively, the pharmaceutical composition ofthe present invention may be freeze-dried so as to realize a dosage formsuch that the pharmaceutical composition is dissolved and reshapedbefore use. For example, an excipient (e.g., sugar alcohol orsaccharides, such as mannitol and dextrose) can be used for freezedrying.

The pharmaceutical composition of the present invention can beformulated according to a conventional technique. Regarding formulation,see Remington's Pharmaceutical Science, latest edition, Mark PublishingCompany, Easton, U.S.A., for example.

The pharmaceutical composition of the present invention can also be usedin combination with an existing antiviral agent, such as interferon andribavirin.

2-2. Method for Administering Pharmaceutical Composition

The pharmaceutical composition containing the inhibitory agent for HCVinfection of the present invention is preferably administered in a unitdosage form. The pharmaceutical composition can be administered viaperoral administration, interstitial administration (e.g., subcutaneousadministration, intramuscular administration, and intravenousadministration), local administration (e.g., transdermaladministration), or transrectal administration. Therefore, dosage formsof the inhibitory agent for HCV infection are preferably appropriate forthe routes of administration. For example, in the case of interstitialadministration, injection that is performed via bloodstream ispreferred, and thus the dosage form in such a case is liquid.

In the case of injection, injection sites are not particularly limited.Examples of injection include intravenous, intraarterial, intrahepatic,intramuscular, intra-articular, intramedullary, intraspinal,intraventricular, trans dermal, subcutaneous, intradermal,intraperitoneal, intranasal, intraintestinal, and sublingual injections.A preferable example thereof is intravascular injection such asintravenous injection or intraarterial injection. This is because thepharmaceutical composition of the present invention can be immediatelyspread throughout the body via bloodstream and intravascular injectionhas relatively low invasiveness, causing little burden on subjects.Alternatively, intrahepatic or hepatoportal injection may be performed.This is because the inhibitory agent for HCV infection can be caused toact directly on sites where HCV is localized.

When the above inhibitory agent for HCV infection is administered, asingle dosage unit thereof preferably contains effective dose by whichthe activity of inhibiting HCV infection can be exhibited. The term“effective dose” as used herein refers to an amount required for theactive ingredient to exhibit its functions; that is, in the presentinvention, an amount of the inhibitory agent, which is required for theagent to inhibit HCV infection, but causes almost no or never causesharmful adverse reaction on a subject to which the inhibitory agent isadministered. The effective dose can be varied depending on variousconditions such as the information of a subject, formulation, and theroute of administration. Examples of the “information of a subject”include the progression degree or the severity of the disease, generalhealth, age, body weight, gender, dietary life, drug sensitivity, thepresence or the absence of a medicament to be used in combination, andtolerance to treatment. The final dosage and effective dose of the aboveinhibitory agent for HCV infection are determined by doctors accordingto the information and the like of individual subjects. Whenadministration of a large dose of the above inhibitory agent for HCVinfection is required in order to obtain the effects of inhibiting HCVinfection, the agent may be administered in divided doses so as toalleviate the burden on a subject.

A specific example of the dosage is as follows. When the inhibitoryagent is administered to a human adult male (body weight: 60 kg) who isat the early phase of the onset of hepatitis C and does not need acombined use of another medicament, the effective dose per day of theinhibitory agent for HCV infection generally ranges from 1 mg to 2000mg, preferably ranges from 1 mg to 1000 mg, and more preferably rangesfrom 1 mg to 500 mg. The dose less than or higher than the above rangesmay also be administered depending on the state of a subject, the routeof administration, or the like.

When the inhibitory agent for HCV infection of the present invention isadministered to a subject, the effective dosage of the antibody of thepresent invention, which is the active ingredient, per administration isselected from within the range of 0.001 mg to 1000 mg per kg bodyweight. Alternatively, the effective dosage can be selected from withinthe range of 0.01 mg to 100000 mg per body of subject, but is notlimited thereto. Also, the inhibitory agent may be administered eitherbefore or after the patient develops clinical symptoms of the disease.

The antibody having activity of inhibiting HCV infection of the presentinvention can be effective against any hepatitis C. For example, theantibody is effective against chronic hepatitis or fulminant hepatitis,and particularly effective against hepatitis C caused by HCV of genotype2a or 1b.

3. Method for Detecting HCV

Further embodiments of the present invention are a method for detectingHCV and a reagent for detecting HCV, which is used for the method.According to the present invention, HCV particles in a sample can bedetected by an immunological detection method using the anti-HCVantibody of the present invention.

The term “sample” as used herein refers to various samples that cancomprise HCV particles or the envelope proteins thereof. Examplesthereof include cultured cells, cultured cell-disrupted solutions,culture solution supernatants, and human samples. Examples of a humansample include: tissue collected from a human (e.g., postoperativetissue); and various human-derived biological samples such as bodyfluids (e.g., blood, serum, blood plasma, urine, spinal fluid, saliva,lymph fluid, and seminal fluid). Preferable examples thereof includeblood, serum, blood plasma, and urine. Also samples in the presentinvention may be not only liquid samples, but also solid samples. Forexample, donor organs resulting from organ transplantation, tissuesection samples, or the like can be used.

“Immunological detection method” of the present invention can beperformed by a known immunological detection method using a labeledantibody, such as ELISA, EIA, fluorescence immunoassay,radioimmunoassay, or luminescence immunoassay, or, a surface plasmonresonance method (SPR method) or Quarts Crystal Microbalance measurement(QCM). Such an immunological detection method using a labeled antibodyis preferably applied.

ELISA is also referred to as enzyme-linked immunosorbent assay, whichinvolves detecting an antigen-antibody reaction with the use of anenzyme-labeled antibody or antigen and the action of the enzyme based onthe color optical density or fluorescence intensity, so as toquantitatively determine a target antigen contained in a trace amount ina sample. Specifically, the method involves immobilizing the anti-HCVantibody or the HCV particles of the present invention onto asolid-phase support, and then enzymatically detecting an immunologicalreaction between the antibody or the like and HCV. Regarding ELISA, seeknown methods (Ed., Japanese Society of Laboratory Medicine, “ClinicalPathology, Extra Edition, Feature, No. 53, Immunoassay for ClinicalExamination—Techniques and Applications—,” The Clinical Pathology Press,1983; Ed., Eiji Ishikawa et al., “Enzyme Immunoassay,” Third Edition,IGAKU-SHOIN, 1987; Ed., Tsunehiro Kitagawa, “Protein Nucleic AcidEnzyme, Separate Volume, No. 31 Enzyme Immunoassay,” KYORITSU SHUPPANCO., LTD, 1987; Ed., Minoru Irie, “Radioimmunoassay,” KodanshaScientific Ltd., 1974; and Ed., Minoru Irie, “Radioimmunoassay 2,”Kodansha Scientific Ltd., 1979). As the above solid-phase support, aninsoluble support in the form of beads, microplates, test tubes, sticks,or test pieces made of material such as polystyrene, polycarbonate,polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride,nylon, polymethacrylate, latex, gelatin, agarose, cellulose, sepharose,glass, metal, ceramics, or magnetic material can be used herein. Theanti-HCV antibody or HCV particles of the present invention can beimmobilized via binding to a solid-phase support by a known method suchas a physical adsorption method, a chemical binding method, or acombination thereof.

As labeling substances for labeling the anti-HCV antibody: peroxidase(POD), alkaline phosphatase, β-galactosidase, urease, catalase, glucoseoxidase, lactate dehydrogenase, amylase, a biotin-avidin complex, or thelike can be used in the case of ELISA; fluorescein isothiocyanate,tetramethylrhodamine is othiocyanate, substituted rhodamineisothiocyanate, dichlorotriazine isothiocyanate, Alexa480,AlexaFluor488, or the like can be used in the case of fluorescenceimmunoassay; and tritium (³H), iodine 125(¹²⁵I), iodine 131(¹³¹I), orthe like can be used in the case of radioimmunoassay. However, theexamples are not limited to them. Also, in the case of luminescenceimmunoassay, an NADH-FMNH₂-luciferase system, a luminol-hydrogenperoxide-POD system, an acridinium ester system, a dioxetane compoundsystem, or the like can be used. Regarding a method for binding alabeled antigen to an antibody, a known method such as a glutaraldehydemethod, a maleimide method, a pyridyl disulfide method, or a periodicacid method can be used in the case of ELISA; and a known method such asa chloramine T method, or Bolton-Hunter method can be employed in thecase of radioimmunoassay.

Also, the immunoassay of the present invention can be performed bymeasuring the generation of immune complex agglutinates resulting fromimmunonephelometry, latex agglutination reaction, latex turbidimetry,hemagglutination reaction or particle agglutination reaction through anoptical method based on transmitted light or scattered light, or througha visual measurement method. In this case, phosphate buffer, glycinebuffer, tris buffer, Good's buffer, or the like can be used as a solventand further a reaction accelerator such as PEG or a non-specificreaction inhibitor may be contained.

A specific example in which the method for detecting HCV of the presentinvention is applied to ELISA is briefly explained below. First, theanti-HCV antibody of the present invention is immobilized on aninsoluble support. In addition, not only 1 type, but also a plurality oftypes of antibody may be immobilized, as long as they can specificallyrecognize HCV particles. Next, a sample that can contain HCV particlesis caused to act on the surfaces of immobilized antibodies, so thatcomplexes of immobilized antibodies and HCV particles are formed on thesurface of the support. Subsequently, the support is sufficiently washedwith a washing solution, so as to remove unbound substances in thesample other than HCV particles. Furthermore, other anti-HCV-labeledantibodies specifically recognizing HCV particles are prepared. Thelabeled antibodies are caused to act on a support to which complexes ofimmobilized antibodies and HCV particles have been bound. Aftersufficient washing using a washing solution, HCV particles existing inthe sample can be detected via detection using the label.

Also, labeled antibodies and a sample containing HCV particles are mixedin advance to form antigen-antibody complexes, so that the complexes canbe caused to act on immobilized antibodies. When antibodies to beimmobilized are labeled with biotin, biotinylated and immobilizedantibodies, a sample containing HCV particles, and antibodies labeledwith a label other than biotin are all mixed together, so as to formantigen-antibody complexes and to cause avidin to act on thesolid-phased support. Thus, antigen-antibody complexes can be detectedvia labeling other than biotinylation.

Test strips for immunochromatography can also be used for theimmunoassay of the present invention. A test strip forimmunochromatography is composed of a sample receiving part comprising amaterial that easily absorbs a sample, a reagent part containing adiagnostic product of the present invention, a development part where areaction product from the sample and the diagnostic product develops, alabeling part where the reaction product that has developed is colored,and a display part to which the colored reaction product develops, forexample. For example, a commercially available diagnostic of pregnancyhas a form similar to such a test strip. The principle of themeasurement method is as described below. First, a sample is added tothe sample-receiving part, and then the sample-receiving part absorbsthe sample and then causes the sample to reach the reagent part.Subsequently, HCV in the sample and the above anti-HCV antibody conductan antigen-antibody reaction at the reagent part, and then the reactioncomplex migrates in the development part to reach the labeling part. Atthe labeling part, a reaction takes place between the above reactioncomplex and a labeled secondary antibody. When the product resultingfrom the reaction with the labeled secondary antibody develops andreaches the display part, color development is observed. The above teststrip for immunochromatography has extremely low invasiveness, providingno pain or risk due to the use of the reagent to users. Hence, the teststrip can be used for monitoring in the home. Furthermore, the resultscan be precisely examined and/or treated (e.g., surgical excision) atthe level of each medical institution, resulting in prevention ofmetastases and/or recurrences. Such a test strip is also advantageous inthat it can be produced in large amounts at low cost.

According to the embodiments of the present invention, HCV particlesand/or the envelope proteins thereof in a sample can be detected.Therefore, HCV infection via a donor's blood or organ can be prevented.

4. Kit for Detecting HCV

Another embodiment of the present invention is a kit for detecting HCVaccording to “3. Method for detecting HCV” above, which contains theanti-HCV antibody of the present invention. The kit for detecting HCV ofthe present invention can contain a labeled secondary antibody, asubstrate required for detection of the label, a positive control or anegative control, a buffer to be used for dilution or washing of asample, and/or instructions.

EXAMPLES

Hereafter, the present invention is described in greater detail withreference to the examples. It should be noted that these examples areprovided for illustrative purposes and the technical scope of thepresent invention is not limited to these examples.

Example 1 Preparation of J6/JFH-1-HCV Particles

cDNA (genomic cDNA) was obtained through reverse transcription of thetotal region of genomic RNA of the HCV JFH-1 strain (genotype 2a)isolated from a fulminant hepatitis patient. The thus obtained cDNA wascloned into a site downstream of a T7 RNA promoter sequence of a pUC19plasmid. The obtained plasmid DNA (pJFH-1) was prepared according to themethod described in Wakita, T. et al., Nat. Med., 11 (2005) p. 791-796and International Patent Publication WO 2004/104198. The nucleotidesequence of genomic cDNA derived from the JFH-1 strain, which wasinserted in pJFH-1, is as shown in SEQ ID NO: 1. The pJFH-1 was digestedwith EcoR I, and then partially digested with Bcl I. Thus, a plasmid DNAfragment was prepared by excising a fragment (about 2840 bp) rangingfrom the EcoR I site to the first Bcl I site, and then the plasmid DNAfragment was purified.

Meanwhile, HCV genomic cDNA derived from the J6CF strain (GenBankAccession No. AF177036, Yanagi, M., et al., Virology 262: 250-263(1999)) was cloned into a site downstream of a T7 RNA promoter sequenceof a pUC19 plasmid. The thus obtained plasmid DNA (pJ6CF) was preparedaccording to the method described in International Patent Publication WO2006/022422. The pJ6CF was partially digested with EcoR I and Bcl I, thethus obtained about 2840-bp fragment was purified, and then the fragmentwas ligated to the pJFH-1 plasmid DNA fragment prepared by excision ofthe above EcoR I-Bcl I fragment, so that a plasmid DNA (pJ6/JFH-1) wasobtained. DNA (SEQ ID NO: 2) cloned in the pJ6/JFH-1 is chimeric HCVgenome cDNA in which the 5′ untranslated region, the core sequence, theE1 sequence, the E2 sequence, and the p7 sequence, a sequence encodingthe region ranging from the N-terminus to the 16^(th) amino acid residueof the NS2 protein derived from the genomic cDNA of the J6CF strain, anda sequence encoding the region ranging from the 17^(th) amino acidresidue to the C-terminus of the NS2 protein, the NS3 sequence, the NS4Asequence, the NS4B sequence, the NS5A sequence, the NS5B sequence, andthe 3′ untranslated region derived from the genomic cDNA of the JFH-1strain are linked in this order.

The thus prepared pJ6/JFH-1 was cleaved with Xba I, and then Mung BeanNuclease 20U (total amount of reaction solution: 50 μL) was addedthereto, followed by 30 minutes of incubation at 30° C. Thus, the XbaI-cleaved end was blunt-ended. Phenol chloroform extraction and ethanolprecipitation were performed, so that an Xba I-cleaved fragment, fromwhich 4 bases (CTAG) at the cohesive end had been removed, was obtained.RNA was synthesized using the thus cleaved plasmid as a template and aMEGAscript T7 kit (Ambion) (see WO 2006/022422). The thus synthesizedHCV genomic RNAs were each used for introduction into cells, asdescribed below.

Huh7 cells (3×10⁶ cells) and 5 μg of each HCV RNA were suspended in aCytomix solution (120 mM KCl, 0.15 mM CaCl₂, 10 mM K₂HPO₄/KH₂PO₄, 25 mMHepes, 2 mM EGTA, 5 mM MgCl₂, 20 mM ATP, 50 mM glutathione (400 μL)) andthen the suspension was transferred to a 4-mm cuvette. HCV RNA waselectroporated into Huh7 cells using a Gene Pulser (BioRad) at 260 V and950 μF. Thereafter, host cells for introduction of HCV genomic RNA wereseeded in a 10 cm² dish, and then subcultured. During subculture, HCVcore protein contained in culture supernatants was quantitativelydetermined using an HCV antigen ELISA test kit (Ortho ClinicalDiagnostics), and thus production of HCV particles was confirmed.Culture supernatants in which the amounts of the core protein were highand the activity of producing HCV particles was high were selected andthen stored as virus stocks.

The above obtained J6/JFH-1 virus stocks (4×10⁴ ffu/ml) were added in anamount of about 100 μL each to Huh7 cells cultured with 10% FCS-DMEMmedium (containing 1% MEM nonessential amino acid solution (Invitrogen),10 mM HEPES-Tris (pH 7.3), and 1 mM sodium pyruvate) in a 10-cm dish, sothat Huh7 cells were infected with HCV virus.

The cells were adequately subcultured to prevent cells from becomingconfluent, and culture expansion was carried out in 225 cm² flasks fromone flask to 4 flasks and then 12 flasks. Subsequently, cells weredetached from 8 of such 225-cm² flasks, and seeded in two 5-layerCellstacks (registered trademark) (Corning), and a medium was addedthereto in an amount of 650 mL/cellstack. The cells obtained from theremaining 4 flasks were seeded in 12 flasks and thus virus productionwas effectively continued.

On the day following subculture, the media were discarded and 650 mL of2% FCS-DMEM (containing 1% MEM non-essential amino acid solution(Invitrogen), 10 mM HEPES-Tris (pH 7.3), 1 mM sodium pyruvate) wasadded. The media were recovered 3 days after medium exchange, passedthrough a 0.45-μm filter, and the resultants were stored in a deepfreezer. Also, 650 mL of the above 2% FCS-DMEM was added to theCellstacks after the culture supernatants had been recovered, andculture was continued. A similar procedure was repeated 2 days aftermedium exchange and the culture supernatants were recovered. A similarprocedure was then repeated one more time. The culture supernatantsrecovered herein were used in Example 2 below.

As described above, cells, into which chimeric HCV genome RNAsynthesized from pJ6/JFH-1 had been introduced, produced infectious HCVparticles and the supernatant contained the thus produced infectious HCVparticles mixed therein.

Example 2 Purification of J6/JFH-1 HCV Particles

Viral particles produced in Example 1 were purified using the following3-stage process.

(1) Concentration and Diafiltration

With the use of Hollow Fiber Cartridge (GE Healthcare: 500 kDa cut-off,Model No. UFP-500-C-8A, hereinafter, referred to as “Hollow Fiber”), theculture supernatant containing the HCV particles obtained in Example 1above was concentrated 60-fold.

(2) Density-Gradient Ultracentrifugation

To the Ultra-clear 25×89 mm centrifuge tube ((Beckman Coulter, Inc.,Catalog No. 344058), 3 mL of TNE buffer (10 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1 mM EDTA) containing cold 60% sucrose was added, and 7 mL of TNEbuffer containing 20% sucrose was overlaid thereon. Further, 25 ml ofthe sample was overlaid onto the TNE buffer containing 20% sucrose.Ultracentrifugation was carried out using a SW-28 (Beckman Coulter) at28,000 rpm for 4 hours at 4° C.

The bottom of the tube was perforated using the 25 G injection needle(Terumo) and 12 fractions (1 mL each) were obtained. The specificgravity of the solution of each fraction was measured, so that theformation of sucrose density gradient was confirmed. The fractions withthe 3^(rd), the e^(h), and the 5^(th) highest specific gravity wererecovered in descending order of their specific gravity, and then usedfor concentration and buffer exchange.

(3) Concentration and Buffer Exchange

The elution fraction was subjected to buffer exchange and concentrationusing Amicon Ultra-15 Centrifugal Filter Units (molecular weight to beeliminated: 100 kDa, Millipore) and TNE buffer. The thus-obtainedconcentrate was used as a virus solution containing infectious HCVparticles in the immunization step described below.

Example 3 Inactivation of HCV

HCV in the virus solution obtained in Example 2 above was inactivatedvia ultraviolet irradiation. As a source of ultraviolet rays, GL-15(Toshiba) was used. The solution containing purified HCV particleshaving an infectious titer of 1×10⁶ ffu/mL was introduced into asilicon-coated polyethylene Eppendorf tube (Assist Co., Ltd,), the tubewas placed at a distance from the source of ultraviolet rays, so thatthe ultraviolet rays would be applied at the intensity of 20 mW/cm², andUV-C was applied for 5 minutes.

After ultraviolet irradiation, HCV particles were serially diluted50-fold, 250-fold, 1.250-fold, 6.250-fold, 31.250-fold, 156.250-fold,and 781.250-fold in DMEM.

On the previous day, the Huh7 cells were seeded on a 96-wellpoly-L-lysine-coated plate (Corning 96 Well Clear Flat BottomPoly-L-Lysine Coated Microplate, Corning) at 1×10⁴ cells/well, theserially-diluted virus particles were seeded thereonto, and culture wasconducted at 37° C. for 72 hours.

After the culture supernatant was removed, the plate was soaked inice-cold methanol to fix the cells. Thereafter, methanol was removed viaair drying, and cells were permeabilized with the use of Block Ace(registered trademark) (Dainippon Pharmaceutical Co., Ltd.) containing0.3% Triton (registered trademark)-X 100 (GE Healthcare). HCV-infectedcells were detected using the clone 2H9 anti-HCV-core antibody (Wakita,T. et al., Nat. Med. 11:791-796, 2005) and goat anti-mouse IgG-Alexa488(Molecular Probes), and the number of HCV-infected cells was countedunder a fluorescent microscope (IX-70; Olympus). The infectious titer ofthe ultraviolet-irradiated HCV was confirmed to be the same or below thedetection limit. Inactivated J6/JFH-1-HCV particles, for which completedisappearance of infectivity had been demonstrated, were used foradministration to mice in the Examples below.

Example 4 Immunization of Mice with Inactivated HCV Particles

An adjuvant for animals, MPL+TDM (Sigma: Sigma Adjuvant System, CatalogNo. 56322) was used as an adjuvant. To 100 μl of a solution containinginactivated J6/JFH-1-HCV particles (equivalent to 2 pmol of the HCV coreprotein) described in Example 3, the equivalent amount of MPL+TDM wasadded to generate an emulsion. Generation of an emulsion was confirmedby preparing an adequate amount of water in a beaker, adding a drop ofthe relevant mixture on the liquid surface, and observing that theliquid would not disperse. Balb/c mice (7-week-old, females) wereetherized, and the emulsion containing inactivated J6/JFH-1-HCVparticles thus prepared was intraperitoneally administered thereto toimmunize the mice.

Two weeks later, to 100 μl of a solution containing J6/JFH-1-HCVparticles (equivalent to 2 pmol of the HCV core protein), the equivalentamount of MPL+TDM was added to generate an emulsion, and then mice werefurther immunized with the emulsion via intraperitoneal administrationas described above. The emulsion was further administeredintraperitoneally to mice 4 weeks later and 6 weeks later for furtherimmunization.

Example 5 Measurement of Activity of Inhibiting HCV Infection in BloodSerum Derived from Mouse Immunized with Inactivated HCV Particles (1)Preparation of Infectious HCV-Like Particles (HCVpp)

HCVpp was prepared according to the method of Bartosch et al. (document:Bartosch, B. et al. (2003) J. Exp. Med., 197, 633-642). This methodinvolves introducing 3 types of expression vector (a vector forexpression of retrovirus Gag-pol, a vector for expression of HCVenvelope proteins, and a retrovirus packaging vector for expression of areporter gene) into animal cells for expression, packaging the reportergene, and thus preparing a pseudo virus expressing the HCV envelopeproteins on the viral surface.

pcDNA J6dC-E2 was used for preparing HCVpp having envelope proteins ofgenotype 2a. The plasmid is an expression vector constructed by cloninga nucleic acid encoding the 132″ to the 750^(th) amino acid residues (apart of the core protein, the E1 protein, and the E2 protein) of theprotein of the J6CF strain that is an HCV strain of genotype 2a (NCBIProtein Accession No. AAF01178.1) into pcDNA3.1.

Gag-Pol 5349 was used as an expression vector constructed by cloning thegenes encoding gag and pol of a mouse leukemia virus thereinto. Luc126was used as a retrovirus packaging vector constructed by cloning aluciferase gene thereinto.

293T cells were subcultured in 10% FCS-DMEM (containing 1% MEMnonessential amino acid solution (Invitrogen), 10 mM HEPES (pH 7.3), 1mM sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin(Gibco: Catalog No. 15140-122)) (hereinafter, referred to as“DMEM-10F”). Collagen-coated flasks (IWAKI: Catalog Nos. 4100-010 and4160-010) were used for culture. 293T cells were seeded incollagen-coated 10-cm dishes (IWAKI: Catalog No. 4020-010) so that theconcentration would be 2.5×10⁶ cells/dish, and then cells were culturedovernight. Opti-MEM (Gibco: Catalog No. 11058), FuGENE6, and 3 types ofconstruct (HCV envelope protein expression vectors pcDNA J6dC-E2,Gag-Pol 5349, and Luc126) were mixed in amounts below. Specifically, 500μl of Opti-MEM, 21.6 μL, of FuGENE6, 1 μg of pcDNA J6dC-E2, 3.1 μg ofGag-Pol 5349, and 3.1 μg of Luc126 were mixed (or mixed at the samemixing ratio), and then incubated at room temperature for 15 minutes.Each medium for 293T cells was exchanged with Opti-MEM (7.5 mL), DNAcomplex was added thereto, and then incubation was performed at 37° C.and 5% CO₂ for 6 hours. After completion of reaction, cells were washedonce with PBS, DMEM-10F (8 mL) was added, and then incubation wasperformed at 37° C. and 5% CO₂ for 48 hours. After completion ofculture, supernatants were recovered, and then filtered with a 0.45-μmfilter, so that an HCVpp solution was obtained. The HCVpp solution wasdispensed 1 mL each and then stored at −80° C.

The thus obtained infectious HCV-like particles having structuralproteins of the J6CF strain of genotype 2a are referred to as “J6CFHCVpp.”

(2) Measurement of Activity of Inhibiting HCV Infection

Normal mouse serum before administration of HCV particles, and 200 μL ofserum from blood collected on day 49 after primary immunization of amouse with inactivated HCV particles (J6/JFH-1-HCV particles) asdescribed in Example 4 were separately applied to 1 mL of a protein Gsepharose column (GE Healthcare). After the columns were washed withPBS, the samples were eluted with 0.1 M glycine buffer (pH 3.0) inamounts of 1 mL/fraction. Immediately after elution, 1 M Tris-HCl (pH9.5) was added to return the pH to neutral. The activity of inhibitingHCV infection of the fraction was measured for the peak fraction of theeluted protein as an IgG fraction. Specifically, the IgG fraction samplewas diluted with DMEM (DMEM containing 10% FCS (invitrogen), 1% MEMnonessential amino acid solution (Invitrogen), 10 mM HEPES-Tris (pH7.3), 1 mM sodium pyruvate) so that the final IgG concentration would be10, 30, 100, and 200 μg/mL. Then the dilutions were separately added tothe J6CF HCVpp solution obtained via the process of “(1) Preparation ofinfectious HCV-like particles (HCVpp)” in the Examples. Incubation wasperformed at 37° C. for 30 minutes. Media of Huh7.5.1 cells (cultured onthe previous day on 96-well plates at 1×10⁴ cells/well) were discarded,IgG fraction samples were added, and then incubation was performed. Theresulting virus solution was added at 50 μL/well and then incubation wasperformed at 37° C. for 3 hours. After the virus solution was discarded,washing was performed once with PBS at 100 μL/well, 200 μL/well mediumwas added and then incubation was performed at 37° C. for 72 hours.After medium was discarded, 25 μL/well DMEM (no serum had been added)and 25 μL/well Steady-Glo (Promega: Catalog No. E2520) were added, andthen cells were lysed according to the instructions included therewith.The cell lysis solution (40 μL/well) was transferred to a white 96-wellplate (Sumitomo Bakelite Co., Ltd.: Catalog No. MS-8496W) and thenluminescence intensity was measured using ARVO X4 (PerkinElmer).

The results are shown in FIG. 1. The vertical axis in FIG. 1 indicatesluciferase activity represented by luminescence intensity and thenumerical values indicated by the horizontal axis are concentrations(μg/mL) of IgG mixed with the J6CF HCVpp solution. Also, “Control”indicates a positive control to which no antibody was added, and “Normalmouse mIgG” indicates a control antibody. The value obtained when theJ6CF HCVpp solution had been mixed with DMEM was shown as a controlindicating 100% infection. The lower the luminescence intensity showsthe higher the activity of inhibiting infection. The IgG fractionderived from the serum of a mouse to which J6/JFH-1-HCV particles hadbeen administered was observed to exhibit the activity of inhibiting HCVinfection in a dose-dependent manner.

Example 6 Preparation of Hybridoma

A mouse myeloma cell line SP2/0 (obtained from ECACC) was cultured inDMEM (Invitrogen) containing 5×10⁻⁵M 2-mercaptoethanol, 100 units/mLpenicillin, 100 μg/mL streptomycin, and 10% FCS (Invitrogen), so thatSP2/0 cells in the logarithmic growth phase were obtained. The cellswere washed 3 times with serum-free DMEM.

Next, spleen cells were extracted from a mouse to which HCV particles(J6/JFH-1-HCV particles) had been administered in Example 4, and thenwashed 3 times with serum-free DMEM. SP2/0 cells and mouse spleen cellswere added to a 50-mL centrifugal tube at a ratio of 1:5, followed by 10minutes of centrifugation at 1,000 rpm. The supernatant was completelyaspirated off, and the bottom of the tube was tapped with fingers toloosen the cell pellet. 1 mL of 50% PEG-1500 solution (Roche) heated at37° C. was added to the cells for 1 minute, and the reaction was allowedto continue at 37° C. for 1 minute. Subsequently, 1 mL of serum-freeDMEM was gradually added for 1 minute, and 1 mL of serum-free DMEM wasgradually added again for 1 minute. In the end, 7 mL of serum-free DMEMwas added for 3 minutes to dilute the PEG solution. The cells in thediluent were centrifuged at 1,000 rpm for 10 minutes, 50 mL of HT medium(DMEM containing 5×10⁻⁵M 2-mercaptoethanol, 100 units/mL penicillin, 100μg/mL streptomycin, 10% FCS, 10⁻⁴M hypoxanthine, and 1.5×10⁻⁵Mthymidine) was added thereto, and the cell pellet was loosened viapipetting. The cells were transferred to two 75-cm² flasks and culturedat 37° C. in a 5% CO₂ incubator overnight.

The cells were centrifuged at 1,000 rpm for 10 minutes and recovered.The cell pellet was loosened via tapping and suspended in 10 mL of DMEM.The cell suspension was added to and thoroughly mixed in 90 mL ofmethylcellulose HAT selective medium (Stem Cell Technology), theresultant was added to 10-cm dishes in amounts of 10 mL per dish, andculture was conducted at 37° C. in a 5% CO₂ incubator.

After the culture for 10 to 14 days, hybridoma colonies, each of whichwas considered to have grown from a single cell, were suctioned with apipette chip, and introduced into each well of a 96-well plate, to which200 μL of HT medium containing 10% hybridoma growth factors (Bio Veris)had been added, and culture was then conducted.

Example 7 Screening of Hybridomas Producing HCV-Infection-InhibitingAntibody

When the hybridomas prepared in Example 6 had sufficiently proliferated,the culture supernatants were recovered, and screening was carried outas follows.

Screening was carried out by immobilizing the E1 and E2 proteins on aplate, evaluating whether or not the antibodies in the hybridomasupernatant would bind to the proteins via EIA, and evaluating whetheror not the antibodies in the hybridoma supernatant would be able toinhibit HCV infection (described in Example 8).

(1) Preparation of E1 Protein and E2 Protein Derived from the J6CFStrain

The E1 and E2 proteins of the J6CF strain were prepared as follows. Withthe use of genomic cDNA derived from the J6CF strain of genotype 2a(GenBank Accession Number AF177036) as a template, a gene encoding theE1 protein lacking a transmembrane region, which is equivalent to aregion ranging from the 192^(nd) to the 352^(nd) amino acid residueswhen the initiator methionine at the N terminus of the full-lengthprotein sequence of J6CF (i.e., the continuous protein sequence encodedby the genome sequence of the J6CF strain: the amino acid sequence underGenBank Accession Number AF177036) was regarded as the 1^(st) amino acidresidue, was amplified via PCR using the Advantage GC2 PCR kit (TakaraBio) with J6E1dTM-s (SEQ ID NO: 3: CACAAGCTTGCCGAAGTGAAGAACATCAGT) andJ6E1dTM-as (SEQ ID NO: 4: GCTCTAGATTAATGAGCCCCGCTAATGATGTC). Theamplified DNA fragment was cloned into pCR-TOPO (Invitrogen), and 3clones were subjected to nucleotide sequence analysis. A clonecontaining an insert having a correct nucleotide sequence was designatedas pTOPO-J6E 1dTM.

pTOPO-J6E1dTM was digested with Hind III and Xba I and a gel containingthe resultant DNA fragment of approximately 500 bp (the E1 fragment) wasexcised. The DNA fragment was purified from the gel using GeneElute(SIGMA). Similarly, p3xFLAG-CMV-9 (SIGMA) was digested with Hind III andXba I, the resultant was electrophoresed on 1% agarose gel, a gelcontaining a fragment of approximately 6,400 bp was excised, and the DNAfragment was purified from the gel using GeneElute (SIGMA). The purifiedDNA fragments were ligated to each other using T4 ligase (Takara Bio),thereby obtaining a CMV-3×FLAGJ6E1dTM animal cell expression vector intowhich the E1 fragment of the J6CF strain had been incorporated.

Subsequently, a gene encoding the E2 protein lacking a transmembraneregion, which is equivalent to a region ranging from the 384^(th) to the720^(th) amino acid residues when the initiator methionine at the Nterminus of the full-length protein sequence of the J6CF strain wasregarded as the 1^(st) amino acid residue, was amplified via PCR usingthe Advantage GC2 PCR kit (Takara Bio) with J6E2dTM-s (SEQ ID NO: 5:CACAAGCTTCGCACCCATACTGTTGGGG) and J6E2dTM-as (SEQ ID NO: 6:GCTCTAGATTACCATCGGACGATGTATTTTGT). The amplified DNA fragment was clonedinto pCR-TOPO (Invitrogen), and 3 clones were subjected to nucleotidesequence analysis. A clone containing an insert having a correctnucleotide sequence was designated as pTOPO-J6E2dTM.

Subsequently, DNA obtained by digesting p3xFLAG-CMV-9 (SIGMA) with HindIII and Xba I was ligated to the DNA fragment of approximately 1,000 bpexcised from pTOPO-J6E2dTM with Hind III and Xba I with the aid of T4DNA ligase for cyclization. The resulting vector was designated asCMV-3×FLAGJ6E2dTM.

CMV-3×FLAGJ6E1dTM or CMV-3×FLAGJ6E2dTM was introduced into the COS1cells derived from monkey kidney cells (Accession Number RCB0143,obtained from Riken Cell Bank) as follows to express the E1 or E2proteins therein.

The COS1 cells were cultured in DMEM (Invitrogen) containing 10% FCS(Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate.The COS1 cells were seeded in a 150-cm² culture flask (Corning Coaster)at a split ratio of 1:2 on the previous day of gene introduction, andthe cells were cultured at 37° C. in a 5% CO₂ incubator overnight.

Separately, DEAE dextran (Pharmacia) and chloroquine (SIGMA) were addedto DMEM (Invitrogen) to final concentrations of 400 μg/ml and 100 μM,respectively, 500 μL (50 μg) of the expression vector (CMV-3×FLAGJ6E1dTMor CMV-3×FLAGJ6E2dTM) was added at 0.1 μg/μL to 13 mL of said solution,and culture was then conducted (the solution was designated as a DEAEdextran-DNA mixture). Subsequently, the supernatant of the cultured COS1cells was aspirated off, and 10 mL of PBS(−) (Nissui) was added theretoand the cells were washed once. After PBS(−) was aspirated off, 13 mL ofthe DEAE dextran-DNA mixture was added thereto per 150-cm² flask, andincubation was then carried out at 37° C. in the presence of 5% CO₂ for4 hours.

4 hours later, the DEAE dextran-DNA mixture was aspirated off, and thecells were washed once with 10 mL of PBS. CHO-SFM medium (Invitrogen)was added at 50 mL per flask, and culture was conducted at 37° C. in thepresence of 5% CO₂. After 4 days, the culture supernatant was collectedin a 50-mL centrifuge tube (Corning Coaster). The collected supernatantwas centrifuged at 6,000 rpm (with the use of a HITACHI RPR9-2 rotor)for 30 minutes at 4° C. and filtered through a 0.2-μm filter (Whatman).

The culture supernatant was purified with the use of anti-FLAG M2agarose (SIGMA) as follows. To 500 mL of the culture supernatant, 1 mLof anti-FLAG M2 agarose was added, and the reaction was allowed toproceed at 4° C. (in a low-temperature chamber) while undergoingagitation in a spinner bottle. After 2 hours, a mixture of thesupernatant and anti-FLAG M2 agarose was transferred to the Econo-Column(BIO-RAD), and flow-through fractions were collected. Subsequently, thecolumn was washed twice with 10 mL of TBS (50 mM Tris-HCl, 150 mM NaCl,pH 7.4). Six fractions (1 mL of each fraction) were eluted with the useof 0.1 M glycine-HCl (pH 3.5). Immediately after elution, 1M Tris-HCl(pH 9.5) was added for neutralization. The fractions (20 μL each) weresubjected to SDS-polyacrylamide gel electrophoresis under reductiveconditions and stained with Coomassie brilliant blue. As a result, theE1 protein or E2 protein derived from the J6CF strain was confirmed tobe purified.

(2) Preparation of Plate with E1 Protein and E2 Protein ImmobilizedThereon

The E1 and E2 proteins derived from the J6CF strain were each dilutedwith PBS to 1 μg/mL. The mixed protein solution (50 μl) of the E1 and E2proteins was added to each well of the immunoplate (NUNC), the plate wasallowed to stand at 4° C. overnight, and the proteins were immobilizedon the plate. The protein solution was removed, 150 μl of Block Ace(Dainippon Pharmaceutical Co., Ltd.) was added to each well, and theplate was subjected to blocking at room temperature for 4 hours.

These plates were used for screening for the anti-HCV antibody in theculture supernatants of hybridomas, as described below.

(3) Screening of Culture Supernatant of Hybridoma Prepared UsingJ6/JFH-1-HCV Particle as an Antigen

The plate prepared in (2) above upon which the E1 and E2 proteinsderived from the J6CF strain had been immobilized was washed 4 timeswith PBS containing 0.1% Tween 20 (SIGMA), 50 μl of each hybridomasupernatant sample obtained in Example 6 was added to each well, and thereaction was allowed to proceed at room temperature for 1 hour while theplate was shaken with a plate mixer for reaction. After the reaction,the plate was washed 4 times with PBS containing 0.1% Tween 20 (SIGMA),50 μl of the HRP-labeled anti-mouse IgG antibody (SIGMA), which had beendiluted 10.000-fold with PBS containing 0.1% Tween 20, was added to eachwell, and the reaction was allowed to proceed at room temperature for 1hour while the plate was shaken. After the reaction, the plate waswashed 4 times with PBS containing 0.1% Tween 20 (SIGMA), color wasdeveloped using a color development kit for peroxidase (SumitomoBakelite Co., Ltd.), the absorbance at 450 nm was measured usingMulti-Scan (Titer-Tech), and positive clones were selected.

Example 8 Evaluation of the Activity of Inhibiting HCV Infection

A hybridoma supernatant was added to the J6CF HCVpp solution obtainedvia the process of “(1) Preparation of infectious HCV-like particles(HCVpp)” in Example 5 and then incubation was performed at 37° C. for 30minutes. After the removal of the medium from Huh7.5.1 cells cultured onthe previous day on a 96-well plate at 1×10⁴ cells/well, the virussolution incubated after addition of the hybridoma supernatant was addedat 50 μL/well, and then incubation was performed at 37° C. for 3 hours.After the virus solution was discarded, the plate was washed once with100 μL/well of PBS, a medium was added at 200 μL/well, and thenincubation was performed again at 37° C. for 72 hours. The medium wasdiscarded, 25 μL/well of DMEM (serum-free) and 25 μL/well of Steady-Glo(Promega: Catalog No. E2520) were added, and thus cells were lysedaccording to instructions included therewith. The cell lysis solutionwas transferred at 40 μL/well to a white 96-well plate (SumitomoBakelite Co., Ltd.: Catalog No. MS-8496W) and then luminescenceintensity was measured using ARVO X4 (PerkinElmer). Luminescenceintensity when the above test was simultaneously conducted with a virussolution prepared by mixing the J6CF HCVpp solution with DMEM (in anamount equivalent to the hybridoma supernatant) was regarded asrepresenting 100% infection. Therefore, luminescence intensity aftermixing with a hybridoma supernatant was expressed with percentages (%),and thus infection (%) was found.

Two types of samples with low infection (%) were selected as hybridomacell line producing monoclonal antibodies having the activity ofinhibiting HCV infection. These cell lines were cloned with limitingdilution, so that the P18-9E hybridoma cell line producing the P18-9Emonoclonal antibody and the P19-7D hybridoma cell line producing theP19-7D monoclonal antibody were obtained.

The P18-9E hybridoma cell line (Accession No: FERM BP-11263) and theP19-7D hybridoma cell line (Accession No: FERM BP-11264) selected in theExamples were deposited at the International Patent Organism Depositaryof the National Institute of Advanced Industrial Science and Technology(Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan) as of 15Oct., 2009.

These hybridoma cell lines can be appropriately cultured at 37° C. usingmedium prepared by adding 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol,and 10% FCS to DMEM.

Example 9 Analysis of HCV Infection-Inhibiting Monoclonal Antibody

Properties of HCV infection-inhibiting monoclonal antibodies producedfrom the hybridoma cell lines obtained in Example 8 were analyzed asfollows. In addition, a monoclonal antibody produced from the P18-9Ehybridoma cell line (Accession No: FERM BP-11263) was designated as“P18-9E monoclonal antibody,” and a monoclonal antibody produced fromthe P19-7D hybridoma cell line (Accession No.: FERM BP-11264) wasdesignated as “P19-7D monoclonal antibody.”

(1) Antibody Subclass

Mouse antibody subclass was analyzed using the culture supernatants ofhybridomas and a Mouse MonoAB ID KIT (Invitrogen). The result ofanalyzing the isotype of the P18-9E monoclonal antibody demonstratedthat the isotype was IgG2b since the H chain was revealed to be γ2b andthe L chain was revealed to be K. The result of analyzing the isotype ofthe P19-7D monoclonal antibody demonstrated that the isotype was IgG1since the H chain was revealed to be γ1 and the L chain was revealed tobe K.

(2) Purification

Hybridomas were cultured in Hybridoma SFM serum free medium (Invitrogen)until confluent. The culture solution was then collected in acentrifugation tube and then centrifugation was performed at 1500 rpmfor 5 minutes. The culture supernatant was added to Prosep-G (Millipore)and then washed with 20 bed volumes of PBS. Subsequently, 6 fractionswere eluted with 1 bed volume of 0.1 M glycine-HCl (pH 3.0). Immediatelyafter elution, 1 M Tris-HCl (pH 9.5) was added for neutralization. Theabsorbance at 280 nm of each fraction was measured using NanoDrop(NanoDrop Technologies, ND-1000). Fractions (OD280 nm >0.1) containingthe proteins were collected. Buffer exchange with PBS was performedwhile concentrating using Amicon Ultra 50 K (Millipore), and thenfiltration was performed with a 0.22-μm filter. The concentrated sample(20 μL) was subjected to SDS-polyacrylamide gel electrophoresis underreductive conditions and non-reductive conditions, and then stained withCoomassie brilliant blue, so that the protein was confirmed to be IgG.The absorbance at 280 nm of the sample was measured, and then the amountof the antibody contained in the solution (10 mg/mL IgG=13.7 OD (basedon OD)) was calculated.

(3) The Activity of Inhibiting HCV Infection of Purified MonoclonalAntibody A. Activity of Inhibiting HCV Infection Against InfectiousHCV-Like Particles (J6CF HCVpp)

The purified P18-9E monoclonal antibody or the purified P19-7Dmonoclonal antibody was added to the J6CF HCVpp solution obtained viathe process of “(1) Preparation of infectious HCV-like particles(HCVpp)” in Example 5 and then incubation was performed at 37° C. for 30minutes. The purified P18-9E monoclonal antibody and the purified P19-7Dmonoclonal antibody were diluted with PBS so that the finalconcentration would be 100 μg/ml and 300 μg/ml, respectively, and thenused.

Huh7.5.1 cells were subcultured in 10% FCS-DMEM (containing 1% MEMnonessential amino acid solution (Invitrogen), 10 mM HEPES (pH 7.3), 1mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/mL streptomycin(Gibco: Catalog No. 15140-122)).

After medium was discarded from Huh7.5.1 cells cultured on the previousday on a 96-well plate at 1×10⁴ cells/well, the virus solution to whicha purified monoclonal antibody had been added and then incubated wasadded at 50 μL/well. Incubation was performed at 37° C. for 3 hours.After the virus solution was discarded, washing was performed once with100 μL/well of PBS, 200 μL/well medium was added, and then incubationwas performed at 37° C. for 72 hours. After the medium was discarded, 25μL/well of DMEM (serum-free) and 25 μL/well of Steady-Glo (Promega:Catalog No. E2520) were added and then cells were lysed according toinstructions included therewith. The cell lysis solution was transferredat 40 μL/well to a white 96-well plate (Sumitomo Bakelite Co., Ltd.:Catalog No. MS-8496W), and then luminescence intensity was measuredusing ARVO X4 (PerkinElmer). The luminescence intensity when the abovetest was simultaneously conducted with a virus solution prepared bymixing the J6CF HCVpp solution with PBS (in an amount equivalent to thediluted purified monoclonal antibody) was regarded as representing 100%infection. Luminescence intensity after mixing with the purifiedmonoclonal antibody was expressed with percentages (%), and thusinfection (%) was found.

FIG. 2 shows the activity of inhibiting HCV infection exhibited by theP18-9E monoclonal antibody. In FIG. 2, the vertical axis indicatesinfection (%), “PBS” along the horizontal axis indicates the result of apositive control to which no antibody was added, and “P18-9E 100 μg/mL”indicates the final concentration of the P18-9E monoclonal antibodymixed with the J6CF HCVpp solution. As shown in FIG. 2, the purifiedP18-9E monoclonal antibody inhibited infection with J6CF HCVpp.

FIG. 3 shows the activity of inhibiting HCV infection exhibited by theP19-7D monoclonal antibody. In FIG. 3, the vertical axis indicatesinfection (%), “P19-7D 100 μg/mL, 300 μg/mL” along the horizontal axisindicate the final concentrations of the P19-7D monoclonal antibodymixed with the J6CF HCVpp solution. Also, “P.C” indicates the result ofa positive control to which no antibody was added, “IgG” indicates theresult for the mouse IgG (300 μg/mL) as a control antibody. As shown inFIG. 3, the purified P19-7D monoclonal antibody inhibited infection withJ6CF HCVpp.

B. Activity of Inhibiting HCV Infection Against Infectious HCV Particles(J6/JFH1 HCVcc)

The J6/JFH1-HCV particle solution (concentrate of the culturesupernatant containing HCV particles) (hereinafter, referred to as“J6/JFH1 HCVcc”) obtained via the process of (1) of Example 2 above wasused as an HCVcc virus solution.

Huh7.5.1 cells were subcultured in 10% FCS-DMEM (containing 1% MEMnonessential amino acid solution (Invitrogen), 10 mM HEPES (pH 7.3), 1mM sodium pyruvate, 100 units/mL penicillin, 100 μg/mL streptomycin(Gibco: Catalog No. 15140-122)). Huh7.5.1 cells were seeded on a 96-wellplate (poly-D-lysin-coated) at 1×10⁴ cells/well, and then culturedovernight. The J6/JFH1 HCVcc solution was mixed with the purified P18-9Emonoclonal antibody and then incubated at room temperature for 30minutes. At this time, the P18-9E monoclonal antibody was diluted withPBS to 20 μg/mL, 60 μg/mL, or 200 μg/mL and then used (the finalconcentration of the antibody in a mixture was 10 μg/mL, 30 μg/mL, or100 μg/mL). After medium for cells was discarded, the virus solutionincubated after addition of the antibody was added at 50 μL/well, andthen incubation was performed at 37° C. for 3 hours. After the virussolution was discarded, washing was performed once with 100 μL/well ofPBS, DMEM was added at 200 μL/well, and then incubation was performed at37° C. for 72 hours. After removal of the culture supernatant, the platewas soaked in ice-cold methanol, so as to fix the cells. Thereafter,methanol was removed via air drying, a PBS solution containing 3% H₂O₂was added at 100 μL/well, and then incubation was performed at roomtemperature for 5 minutes. After washing twice with 150 μL/well of PBS,Block Ace (registered trademark) (Dainippon Pharmaceutical Co., Ltd.)containing 0.3% Triton (registered trademark)-X 100 (GE Healthcare) wasadded at 100 μL/well, and then blocking was performed at roomtemperature for 1 hour. After washing twice with 150 μL/well of PBS,HRP-labeled anti-HCV-core antibody (Ortho Clinical Diagnostics) diluted100-fold was added at 50 μL/well for reaction to proceed for 1 hour.Subsequently, the plate was washed 4 times with 150 μL/well of PBS, andthen QuantaBlu (registered trademark) (PIERCE) reaction solution wasadded at 50 μL/well. The solution was allowed to stand at roomtemperature for 30 minutes, QuantaBlu stop solution was added at 50μL/well to stop the reaction. The solution was transferred at 20 μL/wellto a black 384-well plate (Corning: Catalog No. 3676). Fluorescenceintensity was measured using ARVO X4 (PerkinElmer). The fluorescenceintensity when the above test was simultaneously conducted with a virussolution prepared by mixing the J6/JFH1 HCVcc solution with PBS (in anamount equivalent to the diluted purified monoclonal antibody) wasregarded as representing 100% infection. Fluorescence intensity aftermixing with the purified monoclonal antibody was expressed withpercentages (%), and thus infection (%) was found.

The results are shown in FIG. 4. In FIG. 4, the vertical axis indicatesinfection (%), “P18-9E 10 μg/mL, 30 μg/mL, 100 μg/mL” along thehorizontal axis indicate the final concentrations of the P18-9Emonoclonal antibody mixed with the J6/JFH1 HCVcc solution. Also, “PBS”indicates the result of a positive control to which no antibody wasadded, “mouse IgG” indicates the result for a negative control antibody,“CD81 0.1 μg/mL” indicates the result for a positive control antibody.As shown in FIG. 4, the purified P18-9E monoclonal antibody inhibitedinfection with J6/JFH1 HCVcc.

These results demonstrated that the P18-9E and the P19-7D monoclonalantibodies are neutralizing antibodies having activity of inhibiting HCVinfection.

(4) Analysis of Epitope of Monoclonal Antibody

Regarding the 1^(st) to the 162″ amino acid residues (SEQ ID NO: 7) ofthe E1 protein in the J6CF strain and the 1^(st) to the 337^(th) aminoacid residues (SEQ ID NO: 8) of the E2 protein in the J6CF strain,peptides consisting of amino acid sequences designed by shifting aminoacid residues three by three from the N terminus of 10 continuous aminoacids were synthesized. The N terminus of each peptide was biotinylated,and the C terminus of the same was glycine amide (the peptides weresynthesized by JPT on consignment).

The synthesized peptides were dissolved in DMSO and then dissolved inPBS at 0.01 nmol/μL. 50 μl of the peptide solution was added to eachwell of the streptavidin-coated plate (Nunc) and the reaction wasallowed to proceed at room temperature for 1 hour. Subsequently, thepeptide solution was discarded, Blocking One (Nacalai Tesque) was addedat 200 μL/well, and the plate was allowed to stand at room temperaturefor 5 hours. The Blocking One solution was discarded, the plate waswashed 4 times with PBS (150 μL/well, pH 7.2) containing 0.05% Tween 20,the monoclonal antibody diluted to 1 μg/mL with PBS (pH 7.2) containing0.05% Tween 20 was added at 50 μL/well, and the reaction was allowed toproceed at room temperature for 1 hour. Subsequently, the antibodysolution was discarded, the plate was washed 5 times with 150 μL/well ofPBS (pH 7.2) containing 0.05% Tween 20, the HRP-labeled anti-mouse IgGgoat antibody (GE Healthcare) diluted 5.000-fold with PBS containing0.05% Tween 20 was added at 50 μL/well, and the reaction was allowed toproceed at room temperature for 1 hour. After the reaction, the antibodysolution was discarded, and the plate was washed 5 times with 150μL/well of PBS (pH 7.2) containing 0.05% Tween 20. Subsequently,antibodies bound to peptides were detected using a color development kitfor HPR (Sumitomo Bakelite Co., Ltd.) and a spectrophotometer (OD 450nm).

As a result, the purified P18-9E monoclonal antibody and the purifiedP19-7D monoclonal antibody did not react with any peptide. The resultsuggested that the P18-9E monoclonal antibody and the P19-7D monoclonalantibody are antibodies recognizing conformational epitopes.

(5) Cloning of DNA Encoding V Region of Monoclonal Antibody

DNA encoding a V region of the mouse monoclonal antibody against HCVparticles was cloned as follows.

Total RNA was prepared from the hybridoma cell line using PureLinkMicro-to-Midi (Invitrogen) in accordance with the method described inmanuals included therewith. Specifically, 2×10⁶ cells of the P18-9Ehybridoma cell line and 2×10⁶ cells of the P19-7D hybridoma cell linewere each suspended in 0.5 mL of RNA lysis solution, and the cells werepassed through a syringe with a 18 gauge needle several times forhomogenization. The resulting homogenate was centrifuged, 70% ethanolwas added to the same amount of the resulting supernatant, and themixture was applied on an RNA spin cartridge. The cartridge wasthoroughly washed with the addition of a washing solution and RNA waseluted with RNase-free water.

With the use of IgG-μ-type H-chain- and IgG-κ-type L-chain-specific 3′primers of Mouse Ig-Primer Set (Novagen), single-stranded cDNA wassynthesized from total RNA. Specifically, to 3 μg of each RNA, 2 pmol ofthe p-type H-chain-specific 3′ primer or the κ-type L-chain-specific 3′primer, and then 1 μL of a 10 mM dNTPs solution were added. Moreover,distilled water was added so that the final volume would be 13 μL,annealing was performed at 65° C. for 10 minutes, and then the solutionwas left to stand on ice. To the solution, 1 μL of 0.1 M DTT, 1 μL ofRNA OUT, 4 μL of 5×RT buffer included with SuperScriptIII (Invitrogen),and 1 μL of reverse transcriptase SuperScriptII (Invitrogen) were addedfor reaction to proceed at 50° C. for 60 minutes and then 70° C. for 15minutes. After stored at 4° C., the solution was directly used for thenext step, polymerase chain reaction (PCR). PCR was performed using aMouse Ig-Primer Set as a primer set for PCR according to the conditionsas in manuals included therewith. PCR was performed using GeneAmp(registered trademark) PCR System 9700 (Applied Biosystems) andAdvantage GC2 DNA polymerase kit (TAKARA). Specifically, foramplification of H chains, PCR was performed using a primer (hybridizingto the leader sequence of mouse p-type H-chain) ranging from MuIgVH5′-Ato MuIgVH5′-F and a MuIgGVH3′-1 primer (hybridizing to mouse p-type CH)included with the kit. For amplification of L chains, PCR was performedusing a primer (hybridizing to the leader sequence of mouse κ-typeL-chain) ranging from MuIgκVL5-′A to MuIgκVL5′-F and a MuIgκVL3′-1primer (hybridizing to mouse κ-type CL) included with the kit. To 1-4 μLof the above-prepared reaction mixture resulting from single-strandedcDNA synthesis, 10 μL of 5×PCR buffer included with the Advantage GC2DNA polymerase kit, 5 μL of GC Melt, 5 μl of 2 mM dNTPs solution, 1 μLof Advantage GC2 DNA polymerase mix, 2-5 pmol of MuIgκVL 5′ primer, and2 pmol of MuIgκVL3′-1 primer were added. Distilled water was furtheradded so that the final volume would be 50 μL. 50 μL of the PCR solutionwas incubated at 94° C. for 3 minutes and then performed a thermal cycleat 94° C. for 1 minute, 60° C. for 1 minute, and 72° C. for 2 minutes,in that order. This thermal cycle was repeated 40 times and the reactionmixture was further incubated at 72° C. for 6 minutes.

For cloning of a DNA fragment amplified by the PCR method as describedabove, a TOPO TA Cloning kit (Invitrogen) was used. The pCR-TOPO vectorincluded with the kit, the DNA fragment, and a salt solution includedwith the kit were added, the mixture was allowed to stand at roomtemperature for 5 minutes, and a part of the reaction solution was addedto competent cells of E. Coli DH5α (TAKARA). The E. Coli competent cellswere placed on ice for 30 minutes, subsequently heated at 42° C. for 45seconds, and placed again on ice for 2 minutes. SOC medium (roomtemperature) was added thereto, the cells were cultured at 37° C. for 1hour and seeded on an agar medium, and then cultured at 37° C.overnight. Plasmid DNA was prepared from the resulting transformant, andthe nucleotide sequence of the cloned DNA was determined in accordancewith a conventional technique.

(6) Analysis of Nucleic Acid Sequence of cDNA Encoding V Region ofMonoclonal Antibody

The nucleotide sequences of which were determined in (5) above and thenDNAs (cDNAs encoding VH and VL, respectively, of the mouse monoclonalantibody) cloned in the plasmids were analyzed as follows.

Regarding the H chains of the P18-9E monoclonal antibody, 6 clonesresulting from cloning of the PCR product amplified with the use of theMuIgVH5′-B 5′ primer were analyzed. As a result, 1 out of the 6 cloneswas observed to have a substitution at one position in the nucleotidesequence, but the 5 clones had the same V region nucleotide sequences.DNA samples from which the 5 clones had been obtained were selected, andthen the nucleotide sequence of the H-chain cDNA of the P18-9Emonoclonal antibody was determined (SEQ ID NO: 9).

Regarding the L chains of the P18-9E monoclonal antibody, 3 clonesresulting from cloning of the PCR product amplified with the use of theMuIgκVL5′-F 5′ primer were analyzed. As a result, 1 out of the 3 cloneswas observed to have substitutions at two positions in the nucleotidesequence, but the 2 clones had the same V region nucleotide sequences.Bases at the 2 substitution positions in the 2 clones were selected, andthen the nucleotide sequence of the L-chain cDNA of the P18-9Emonoclonal antibody was determined (SEQ ID NO: 10).

Regarding the H chains of the P19-7D monoclonal antibody, the nucleotidesequences obtained from 6 clones resulting from cloning of the productamplified with the use of the MuIgVH5′-C 5′ primer were analyzed. As aresult, 1 out of the 6 clones was observed to have substitutions at 3positions in the nucleotide sequence, but the remaining 5 clones had thesame bases at the 3 positions. Bases at the 3 substitution positions inthe 5 clones were selected, and then the nucleotide sequence of theH-chain cDNA of the P19-7D monoclonal antibody was determined (SEQ IDNO: 11).

Regarding the L chains of the P19-7D monoclonal antibody, 4 clonesresulting from cloning of the PCR product amplified with the use of theMuIgκVL5′-G 5′ primer were analyzed. As a result, 1 out of the 4 cloneswas observed to have substitutions at 2 positions in nucleotide sequenceof the V region, but the 3 clones had the same nucleotide sequences.Bases at the 2 positions in the 3 clones were selected, and then thenucleotide sequence of the L-chain cDNA of the P19-7D monoclonalantibody was determined (SEQ ID NO: 12).

Also, through a search of a database for encoded amino acid sequencesdeduced from the nucleic acid sequences, amino acid sequences rangingfrom FR1 to FR4 (J region) of V-regions in H-chain and L-chain of theP18-9E monoclonal antibody and the P19-7D monoclonal antibody werededuced (SEQ ID NOs: 13-16). Further, CDR1 to CDR3 deduced from theamino acid sequences are shown in FIG. 5.

The amino acid sequence of FR1 of the VH of the P18-9E monoclonalantibody is DAQGQMQQSGPELVKPGASVKLSCKTTDFTFN (SEQ ID NO: 17), the aminoacid sequence of CDR1 of the same is RNYIS (SEQ ID NO: 18), the aminoacid sequence of FR2 of the same is WLRQKPGQSLEWIA (SEQ ID NO: 19), theamino acid sequence of CDR2 of the same is WIYAGTGGTKYNQKFTG (SEQ ID NO:20), the amino acid sequence of FR3 of the same isKAQMTVDTSSHTAYMQFSNLTTEDSAVYYCAR (SEQ ID NO: 21), the amino acidsequence of CDR3 of the same is YLFDGYYIPLFDY (SEQ ID NO: 22), and theamino acid sequence of FR4 of the same (also referred to as J region, Jsegment, or J chain) is WGQGTTLTVS (SEQ ID NO: 23).

The amino acid sequence of FR1 of the VL of the P18-9E monoclonalantibody is AQCDVQITQSPSYLAASPGETISINC (SEQ ID NO: 24), the amino acidsequence of CDR1 of the same is RANKSIDKYLA (SEQ ID NO: 25), the aminoacid sequence of FR2 of the same is WYQEKPGKTNKLLIY (SEQ ID NO: 26), theamino acid sequence of CDR2 of the same is SGSTLQS (SEQ ID NO: 27), theamino acid sequence of FR3 of the same isGVPSKFSGSGSGTDFTLTISSLEPEDFAMYYC (SEQ ID NO: 28), the amino acidsequence of CDR3 of the same is QQHNEYPLT (SEQ ID NO: 29), and the aminoacid sequence of the FR4 (J region) of the same is FGAGTKLDLRR (SEQ IDNO: 30).

The amino acid sequence of FR1 of the VH of the P19-7D monoclonalantibody is LSQPSQSLSITCTVSGFSLT (SEQ ID NO: 31), the amino acidsequence of CDR1 of the same is TYGVH (SEQ ID NO: 32), the amino acidsequence of FR2 of the same is WVRQSPGKGLEWLG (SEQ ID NO: 33), the aminoacid sequence of CDR2 of the same is VIWRGGSTDYNAAFLS (SEQ ID NO: 34),the amino acid sequence of FR3 of the same isRLSITKDNSKSQVFFKMNSLQPDDTAIYYCAKN (SEQ ID NO: 35), the amino acidsequence of CDR3 of the same is SWDGAY (SEQ ID NO: 36), and the aminoacid sequence of FR4 (J region) of the same is WGQGTLVTVS (SEQ ID NO:37).

The amino acid sequence of FR1 of the VL of the P19-7D monoclonalantibody is SSSDVVMTQTPLSLPVSLGDQASISC (SEQ ID NO: 38), the amino acidsequence of CDR1 of the same is RSSQSLLHSNGNTYLH (SEQ ID NO: 39), theamino acid sequence of FR2 of the same is WYLQKPGQSPKLLIY (SEQ ID NO:40), the amino acid sequence of CDR2 of the same is KVSNRFS (SEQ ID NO:41), the amino acid sequence of FR3 of the same isGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFC (SEQ ID NO: 42), the amino acidsequence of CDR3 of the same is SQNTHFPWT (SEQ ID NO: 43), and the aminoacid sequence of FR4 (J region) of the same is FGGGTELEISR (SEQ ID NO:44).

The nucleotide sequences and the amino acid sequences of V regions inH-chain and L-chain playing a role in recognition of conformationalepitopes by the P18-9E and the P19-7D monoclonal antibodies wererevealed.

(7) Analysis of Properties of P18-9E Monoclonal Antibody and P19-7DMonoclonal Antibody Using HCV-Like Particles (HCV-VLP) A. Examination ofReactivity of (HCV-VLP) Monoclonal Antibody Against HCV-Like Particles(HCV-VLP) Using Enzyme Immunoassay (EIA) (a) Preparation of HCV-LikeParticles (HCV-VLP)

HCV-like particles (HCV-VLP) are empty particles containing no viralgenome. Empty particles (HCV-VLP) expressing the HCV E1 protein and E2protein on the surfaces can be prepared from 293T cells caused toexpress the HCV E1 protein and E2 protein using a MembranePro(registered trademark) Reagent (Invitrogen).

293T cells were cultured in a medium prepared by adding 50 mL of FCS(GIBCO) and 5 mL of PenStrep (Invitrogen) to 500 mL of DMEM(Invitrogen). 1.2×10⁷ cells of 293T were seeded in a 225 cm²collagen-coated flask (IWAKI), and then cultured for a whole day at 37°C. and 5% CO₂.

Four mL of Opti-MEM I (GIBCO) and 216 μl of Lipofectamine 2000(Invitrogen) were mixed. After 5 minutes of incubation at roomtemperature, 4 mL of Opti-MEM I (GIBCO), 10.8 μg of pcDNA J6dC-E2(expression plasmid for the E1 protein and the E2 protein of the HCVJ6CF strain described in Example 5) and 32.4 μL of MembranePro(registered trademark) Reagent (Invitrogen) were further mixed and thenadded, and then the solution was incuvated at room temperature for 20minutes. The solution was added dropwise to the culture solution of 293Tcells cultured for a whole day, and then the cells were incuvated at 37°C. and 5% CO₂ for 18 hours. Subsequently, the culture solution wasremoved, the medium was exchanged with 35 mL of a fresh medium (fromwhich PenStrep had been removed) and then cells were cultured forfurther 48 hours. The culture supernatant was collected in a 50-mLcentrifugation tube, and then centrifugation was performed at 4° C. and2,000×g for 10 minutes. The supernatant was transferred to a new 50-mLcentrifugation tube (2 mL was left without transfer thereof), and then 7mL of MembranePro Precipitation Mix (Invitrogen) was added. Theresultant was mixed by inversion for several times, and then left tostand for a whole day or longer at 4° C. Thereafter, centrifugation wasperformed at 4° C. and 5,500×g for 5 minutes, and then the supernatantwas removed using a pipette. MembranePro Precipitation Mix was diluted6-fold with 1×PBS(−), 5 mL of the resulting solution was added to thepellet, centrifugation was further performed at 4° C. and 5,500×g for 5minutes, and then the supernatant was removed using a pipette. 500 μL ofPBS(−) was added to the pellet for suspension, the suspension wasdispensed, and then the resultants were stored at −80° C. until use. Theresultants were designated as “HCV-VLP.”

(b) Preparation of E1 Protein and E2 Protein (Recombinant Proteins)Derived from the J6CF Strain

E1 protein and E2 protein derived from the J6CF strain (recombinantproteins) were separately prepared by the method described in (1) ofExample 7.

(c) Enzyme Immunoassay (EIA)

To a 96-well plate (Nunc), a mixture of the E1 protein and the E2protein which are recombinant proteins (50 ng each) was diluted withPBS(−) and then added at 50 μL/well for immobilization. Similarly,HCV-VLP prepared in “a. Preparation of HCV-like particles (HCV-VLP)” wasadded at 500 ng/well, and then the solution was incubated for a wholeday at 4° C. for immobilization. The antigen solution was decanted off,Blocking One (Nacalai Tesque) diluted 5-fold with milliQwater was addedat 200 μL/well, and then the solution was incubated at room temperaturefor 1 hour, so that blocking was performed. The blocking solution wasdecanted off and the resultant was washed twice with 0.05% (v/v) Tween20 (Sigma)-containing PBS(−) (hereinafter, referred to as “washingsolution”). A monoclonal antibody solution (P18-9E or P19-7D) dilutedwith a washing solution was added at 50 μL/well. To negative controlwells, a washing solution alone was added at 50 μL/well. After about 1.5hours of incubation at room temperature, washing was performed 3 timeswith a washing solution. Next, an HRP-labeled anti-mouse IgG antibody(Amersham) diluted 3.000-fold with a washing solution was added at 50μL/well. After 1 hour of incubation at room temperature, the resultantwas washed 4 times with a washing solution. A color development solutionwas prepared according to instructions included with a color developmentkit for peroxidase (Sumiron) and then added at 50 μL/well. After 15minutes of incubation at room temperature, a reaction stop solutionincluded with the kit was added at 50 μL/well. Subsequently, absorbanceat 450 nm was measured using a microplate reader (Model 680, Bio-Rad).The absorbance of the negative control well was subtracted from theabsorbance of each well, and the thus obtained value was designated asrepresenting the reaction of each solution.

In this experiment, the 8D10-3 monoclonal antibody (WO2010/038789) wasused as a control antibody. The 8D10-3 monoclonal antibody was obtainedby immunizing a BALB/c mouse with a recombinant E2 protein, which has alinear epitope of the E2 protein.

The results are shown in FIG. 6 to FIG. 8. FIG. 6 shows the results ofEIA for the 8D10-3 monoclonal antibody with the use of a plate on whicha mixture of a recombinant E1 protein and a recombinant E2 protein hadbeen immobilized. FIG. 7 shows the results of EIA for the 8D10-3monoclonal antibody and the P18-9E monoclonal antibody with the use ofplates on which HCV-like particles (HCV-VLP) were immobilized. FIG. 8shows the results of EIA for the 8D10-3 monoclonal antibody and theP19-7D monoclonal antibody with the use of plates on which HCV-likeparticles (HCV-VLP) were immobilized. Vertical axes in the figuresindicate the values of absorbance at 450 nm and the horizontal axes inthe same indicate the concentrations (μg/mL) of the monoclonalantibodies.

As a result, the 8D10-3 monoclonal antibody reacted with the mixture ofrecombinant proteins, the E1 protein and the E2 protein (FIG. 6). On theother hand, the reaction of the 8D10-3 monoclonal antibody againstHCV-VLP was found to be weaker than that of the P18-9E monoclonalantibody against HCV-VLP (FIG. 7). Similarly, the reaction of the 8D10-3monoclonal antibody against HCV-VLP was weaker than that of the P19-7Dmonoclonal antibody against HCV-VLP (FIG. 8).

Whereas the 8D10-3 monoclonal antibody recognising a linear epitope ofthe E2 protein had weak ability to recognize the envelope structure onthe HCV-VLP surface, the P18-9E monoclonal antibody and the P19-7Dmonoclonal antibody were suggested to be highly reactive to HCV-VLP andrecognize the envelope conformation of a complex of the E1 protein andthe E2 protein on the surfaces of HCV particles.

B. Examination of Reactivity of Monoclonal Antibody Against HCV-LikeParticles (HCV-VLP) Using Biacore (a) Immobilization of Protein A/G

Among sensor chips for surface plasmon resonance measuring apparatusBiacore S51 (GE), Series S sensor chip CM-5 (GE) was mounted on BiacoreS51, HBS-EP (GE), which is assay buffer diluted 1-fold with ultrapurewater, was allowed to run over the chip at a flow rate of 30 μL/min. Theinternal temperature was set at 25° C. EDC(N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrorochloride) andNHS (N-hydroxysuccinimide) in an amine coupling kit (GE) were preparedto be 100 mM and 400 mM, respectively. Protein A/G (50 μg/mL, Pierce)was prepared with Acetate 4.0 (GE). With the use of the thus preparedEDC and NHS, and an ethanolamine solution in the same kit, protein A/Gwas immobilized by amine coupling. A series of immobilization procedureswere performed at a flow rate of 10 μL/min, and the time for addition ofprotein A/G was determined to be 10 minutes. Therefore, protein A/G wasimmobilized in almost equivalent amounts into both spots 1 and 2 of thesame flow cell.

(b) Capture of Antibody

The purified P18-9E monoclonal antibody and mouse IgG (Sigma) adjustedat 20 μg/mL with HBS-EP were allowed to run over the above chip at aflow rate of 10 μL/min for 8 minutes. With the use of affinity ofProtein A/G and the Fc site of each antibody, the P18-9E monoclonalantibody was captured in spot 1 and the mouse IgG was captured in spot 2on the chip.

(c) Association and Dissociation of VLP

HCV-VLP adjusted at 30, 100, or 300 μg/mL with HBS-EP was allowed to runover the chip at a flow rate of 10 μL/min for 3 minutes. Bindingreaction of VLP was monitored by real-time measurement of surfaceplasmon resonance, HBS-EP was allowed to run for 10 minutes and thendissociation reaction was measured.

(d) Analysis

S51 Evaluation was used for analysis. Measured values were expressedwith RU (Resonance Unit; that is the unit of resonance response).Correction was carried out by subtracting a value representing thereaction of HCV-VLP against mouse IgG (regarded as a value representingnon-specific adsorption) from a value representing the reaction ofHCV-VLP against the P18-9E monoclonal antibody.

(e) Result

As a result, a VLP-concentration-dependent manner and antigen-antibodyreaction-like gentle association and dissociation to the P18-9Emonoclonal antibody were observed. The result also suggested that theP18-9E monoclonal antibody binds to HCV-VLP and recognizes envelopeconformation formed by the complex of the E1 protein and the E2 proteinon the surfaces of HCV particles.

INDUSTRIAL APPLICABILITY

The antibody of the present invention having activity of inhibiting HCVinfection can be used as a medicament for treatment and prevention ofHCV infection.

Sequence Listing Free Text

SEQ ID NO: 1 discloses the HCV genome cDNA sequence cloned into pJFH-1.SEQ ID NO: 2 discloses the chimeric HCV genome cDNA sequence cloned intopJ6/JFH-1.SEQ ID NO: 3 discloses the sequence of the primer (J6E1dTM-s).SEQ ID NO: 4 discloses the sequence of the primer (J6E1dTM-as).SEQ ID NO: 5 discloses the sequence of the primer (J6E2dTM-s).SEQ ID NO: 6 discloses the sequence of the primer (J6E2dTM-as).SEQ ID NO: 7 discloses the sequence of the 1^(st) to the 162″ amino acidresidues of the E1 protein of the J6CF strain.SEQ ID NO: 8 discloses the sequence of the 1^(st) to the 337^(th) aminoacid residues of the E2 protein of the J6CF strain.SEQ ID NO: 9 discloses the nucleotide sequence of the gene encoding VHof the P18-9E monoclonal antibody.SEQ ID NO: 10 discloses the nucleotide sequence of the gene encoding VLof the P18-9E monoclonal antibody.SEQ ID NO: 11 discloses the nucleotide sequence of the gene encoding VHof the P19-7D monoclonal antibody.SEQ ID NO: 12 discloses the nucleotide sequence of the gene encoding VLof the P19-7D monoclonal antibody.SEQ ID NO: 13 discloses the amino acid sequence of VH of the P18-9Emonoclonal antibody.SEQ ID NO: 14 discloses the amino acid sequence of VL of the P18-9Emonoclonal antibody.SEQ ID NO: 15 discloses the amino acid sequence of VH of the P19-7Dmonoclonal antibody.SEQ ID NO: 16 discloses the amino acid sequence of VL of the P19-7Dmonoclonal antibody.SEQ ID NO: 17 discloses the amino acid sequence of FR1 in VH of theP18-9E monoclonal antibody.SEQ ID NO: 18 discloses the amino acid sequence of CDR1 in VH of theP18-9E monoclonal antibody.SEQ ID NO: 19 discloses the amino acid sequence of FR2 in VH of theP18-9E monoclonal antibody.SEQ ID NO: 20 discloses the amino acid sequence of CDR2 in VH of theP18-9E monoclonal antibody.SEQ ID NO: 21 discloses the amino acid sequence of FR3 in VH of theP18-9E monoclonal antibody.SEQ ID NO: 22 discloses the amino acid sequence of CDR3 in VH of theP18-9E monoclonal antibody.SEQ ID NO: 23 discloses the amino acid sequence of FR4 (J region) in VHof the P18-9E monoclonal antibody.SEQ ID NO: 24 discloses the amino acid sequence of FR1 in VL of theP18-9E monoclonal antibody.SEQ ID NO: 25 discloses the amino acid sequence of CDR1 in VL of theP18-9E monoclonal antibody.SEQ ID NO: 26 discloses the amino acid sequence of FR2 in VL of theP18-9E monoclonal antibody.SEQ ID NO: 27 discloses the amino acid sequence of CDR2 in VL of theP18-9E monoclonal antibody.SEQ ID NO: 28 discloses the amino acid sequence of FR3 in VL of theP18-9E monoclonal antibody.SEQ ID NO: 29 discloses the amino acid sequence of CDR3 in VL of theP18-9E monoclonal antibody.SEQ ID NO: 30 discloses the amino acid sequence of FR4 (J region) in VLof the P18-9E monoclonal antibody.SEQ ID NO: 31 discloses the amino acid sequence of FR1 in VH of theP19-7D monoclonal antibody.SEQ ID NO: 32 discloses the amino acid sequence of CDR1 in VH of theP19-7D monoclonal antibody.SEQ ID NO: 33 discloses the amino acid sequence of FR2 in VH of theP19-7D monoclonal antibody.SEQ ID NO: 34 discloses the amino acid sequence of CDR2 in VH of theP19-7D monoclonal antibody.SEQ ID NO: 35 discloses the amino acid sequence of FR3 in VH of theP19-7D monoclonal antibody.SEQ ID NO: 36 discloses the amino acid sequence of CDR3 in VH of theP19-7D monoclonal antibody.SEQ ID NO: 37 discloses the amino acid sequence of FR4 (J region) in VHof the P19-7D monoclonal antibody.SEQ ID NO: 38 discloses the amino acid sequence of FR1 in VL of theP19-7D monoclonal antibody.SEQ ID NO: 39 discloses the amino acid sequence of CDR1 in VL of theP19-7D monoclonal antibody.SEQ ID NO: 40 discloses the amino acid sequence of FR2 in VL of theP19-7D monoclonal antibody.SEQ ID NO: 41 discloses the amino acid sequence of CDR2 in VL of theP19-7D monoclonal antibody.SEQ ID NO: 42 discloses the amino acid sequence of FR3 in VL of theP19-7D monoclonal antibody.SEQ ID NO: 43 discloses the amino acid sequence of CDR3 in VL of theP19-7D monoclonal antibody.SEQ ID NO: 44 discloses the amino acid sequence of FR4 (J region) in VLof the P19-7D monoclonal antibody.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. The isolated anti-hepatitis C virus antibody, wherein a heavy chainvariable region contains a complementarity determining region 1(CDR1_(H)), a complementarity determining region 2 (CDR2_(H)), and acomplementarity determining region 3 (CDR3_(H)), comprising respectivelythe amino acid sequences shown in SEQ ID NOs: 32, 34, and 36 in thesequence listing and a light chain variable region contains acomplementarity determining region 1 (CDR1_(L)), a complementaritydetermining region 2 (CDR2_(L)), and a complementarity determiningregion 3 (CDR3_(L)), comprising respectively the amino acid sequencesshown in SEQ ID NOs: 39, 41, and 43 in the sequence listing.
 2. Theisolated anti-hepatitis C virus antibody according to claim 1, which isproduced by the hybridoma cell line under Accession No. FERM BP-11264.3. The isolated anti-hepatitis C virus antibody according to claim 1,which is a humanized antibody.
 4. A hybridoma cell line, the AccessionNo. of which is FERM BP-11264.
 5. An inhibitory agent for infection withhepatitis C virus, which contains the isolated anti-hepatitis C virusantibody according to claim 1 as an active ingredient.